GENES CLONES CODANT DES PROTEINES DE FUSION IG-CD4 ET LEUR EMPLOI

CLONED GENES ENCODING IG-CD4 FUSION PROTIENS AND THE USE THEREOF

Abrégé anglais

The invention relates to a fusion protein which comprises an
immunoglobulin of the IgM, IgG1 or IgG3 immunoglobulin class, wherein the
variable region of the light or heavy chain has been replaced with CD4 or
fragment thereof which is capable of binding to gp120. The invention also
relates to an immunoglobulin-like molecule comprising the fusion protein of
the invention together with an immunoglobulin light or heavy chain. The
invention also relates to a method of treating HIV or SIV infection
comprising administering the fusion proteins or immunoglobulin-like
molecules of the invention to an animal. The invention also relates to assays
for HIV or SIV comprising contacting a sample suspected of containing HIV
or SIV gp120 with the immunoglobulin-like molecule of fusion protein of the
invention, and detecting whether a complex is formed.

Revendications

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THE EMBODIMENTS OF THE INVENTION IN WHICH AN
EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS
FOLLOWS:
1. A gene encoding a fusion protein which is
capable of being secreted comprising 1) the DNA sequence
of extracellular CD4, or fragment thereof which binds to
HIV gp120 when fused to an immunoglobulin chain, and 2)
the DNA sequence of an immunoglobulin heavy chain,
wherein the DNA sequence which encodes at least the
variable region of said immunoglobulin chain has been
replaced with the DNA sequence which encodes
extracellular CD4, or said gp120 binding fragment
thereof.
2. The fusion protein gene of claim 1, wherein the
DNA sequence which encodes said fragment of CD4 comprises
the following DNA sequence.
<IMG>

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<IMG>
or a degenerate variant thereof.
3. The fusion protein gene of claim 1, wherein said DNA
sequence which encodes said fragment of CD4 comprises the following
DNA sequence:
<IMG>

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<IMG>
or a degenerate variant thereof.
4. The fusion protein gene of claim 1, wherein
said immunoglobulin chain is of the class IgM, IgG1 or
IgG3.
5. A gene encoding a fusion protein which is
capable of being secreted comprising 1) the DNA sequence
of extracellular CD4, or fragment thereof which binds to
HIV gp120 when fused to an immunoglobulin chain, and 2)
the DNA sequence of an immunoglobulin light chain,
wherein the DNA sequence which encodes the variable
region of said immunoglobulin light chain has been
replaced with the DNA sequence which encodes
extracellular CD4, or said HIV gp120 binding fragment
thereof.
6. The fusion protein gene of claim 5, wherein the
DNA sequence which encodes said fragment of CD4 comprises
the following DNA sequence:
<IMG>

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<IMG>
or a degenerate variant thereof.
7. The fusion protein gene of claim 5, wherein the DNA sequence
which encodes said fragment of CD4 comprises the following DNA
sequence:
<IMG>

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<IMG>
or degenerate variant thereof.
8. A vector comprising the fusion protein gene of claim 1.

9. The vector of claim 8, having the identifying characteristics

of pCD4H.gamma.1, which has been deposited in E. coli at the ATCC under
the terms of the Budapest Treaty under Accession No. 67611.

10. The vector of claim 8, having the identifying
characteristics of pCD4Mµ, which as been deposited in E. coli at the

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ATCC under the terms of this Budapest Treaty under Accession
No. 67608.
11. The vector of claim 8, having the identifying
characteristics of pCD4Pµ, which has been deposited in E. coli at the
ATCC under the Budapest Treaty under Accession No. 67609.
12. The vector of claim 8, having the identifying
characteristics of pCD4E.gamma.1, which has been deposited in E. coli at the
ATCC under the terms of the Budapest Treaty under Accession No. 67610.
13. A vector comprising the fusion protein gene of claim 5.
14. A host cell transformed with the vector of claim 8.
15. The host cell of claim 14 which expresses an immunoglobulin light
chain together with the expression product of said fusion protein gene
to give an immunoglobulin-like molecule which binds to gp120.
16. A host cell transformed with the vector of claim 13.
17. The host cell of claim 16 which expresses an immunoglobulin heavy
chain together with the expression product of said fusion protein gene
to give an immunoglobulin-like molecule which binds to HIV or SIV
gp120.
18. The host cell of claim 17, wherein said immunoglobulin heavy
chain is of the immunoglobulin class IgM, IgG1 or IgG3.

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19. A method of producing a fusion protein encoded by a gene of claim 1, said
method
characterized by:
cultivating in a nutrient medium under protein-producing conditions, a host
strain
transformed with a vector comprising the gene of claim 1, said vector further
comprising
expression signals which are recognized by said host strain and direct
expression of said fusion
protein, and
recovering the fusion protein so produced.
20. The method of claim 19, wherein said host strain is a myeloma cell line
which produces
immunoglobulin light chains and said fusion protein comprises an
immunoglobulin heavy chain
of the class IgM, IgG1 or IgG3, wherein an immunoglobulin-like molecule
comprising said
fusion protein is produced.
21. A method of producing a fusion protein which is
capable of being secreted comprising extracellular CD4,
or fragment thereof which binds to gp120 when fused to an
immunoglobulin chain, and an immunoglobulin light chain,
wherein the variable region of the immunoglobulin chain
has been substituted with extracellular CD4, or said
fragment thereof which binds to HIV or SIV gp120, which
comprises:
cultivating in a nutrient medium under protein-producing
conditions, a host strain transformed with the
vector of claim 13, said vector further comprising
expression signals which are recognized by said host
strain and direct expression of said fusion protein, and
recovering the fusion protein so produced.
22. The method of claim 21, wherein said host produces immunoglobulin
heavy chains of the class IgM, IgG1 and IgG3 together with
said fusion protein to give an immunoglobulin-like molecule which
binds to HIV-gp120.

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23. A fusion protein encoded by a gene according to claim 1.
24. The fusion protein CD4H.gamma.1, which is produced by the vector of ATCC
67611.
25. The fusion protein CD4Mµ, which is produced by the vector of ATCC
67609.
26. The fusion protein CD4Pµ, which is produced by the vector of ATCC
67608.
27. The fusion protein CD4E.gamma.1, which is produced by the vector of ATCC
67610.
28. The fusion protein CD4B.gamma.1 according to Table 5.
29. The fusion protein of claim 23 which is detestably labeled.
30. The fusion protein of claim 23, further comprising a
therapeutic agent, radiolabel or NMR imaging agent linked to said
fusion protein.
31. A immunoglobulin-like molecule, comprising the fusion
protein of claim 23 and an immunoglobulin light chain.
32. The immunoglobulin-like molecule of claim 31, further
comprising a detectable label.
33. The immunoglobulin-like molecule of claim 31, further
comprising a therapeutic agent, radiolabel or NMR imaging agent linked
to said immunoglobulin-like molecule.

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34. A fusion protein comprising CD4, or fragment thereof which
binds to HIV gp120, fused at the C-terminus to a second protein
comprising an immunoglobulin light chain where the variable region has
been deleted.
35. The fusion protein of claim 23, wherein said CD4 fragment
comprises the following amino acid sequence:
M N R G
V P F R H L L L V L Q L A L L P A A T Q
G K K V V L G K K G D T V E L T C T A S
Q K K S I Q F H W K N S N Q I K I L G N
Q G S F L T K G P S K L N D R A D S R R
S L W D Q G N F P L I I K N L K I E D S
D T Y I C E V E D Q K E E V Q L L V F G
L T A N S D T H L L Q.
36. The fusion protein of claim 23, wherein said CD4 fragment
comprises the following amino acid sequence:
M N R G
V P F R H L L L V L Q L A L L P A A T Q
G K K V V L G K K G D T V E L T C T A S
Q K K S I Q F H W K N S N Q I K I L G N
Q G S F L T K G P S K L N D R A D S R R
S L W D Q G N F P L I I K N L K I E D S
D T Y I C E V E D Q K E E V Q L L V F G
L T A N S D T H L L Q G Q S L T L T L E
S P P G S S P S V Q C R S P R G K N I Q
G G K T L S V S Q
37. The fusion protein of claim 34 which is detectably labeled.
38. The fusion protein of claim 34, further comprising a

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therapeutic agent, radiolabel or NMR imaging agent linked to said
fusion protein.
39. An immunoglobulin-like molecule comprising the fusion
protein of claim 34 and an immunoglobulin heavy chain of the class
IgM, IgG1 or IgG3.
40. The immunoglobulin-like molecule of claim 39, further
comprising a detectable label.
41. The immunoglobulin-like molecule of claim 39, further
comprising a therapeutic agent, radiolabel or NMR imaging agent linked
to said immunoglobulin-like molecule.
42. A complex comprising the fusion protein of claim 23 and HIV
or SIV gp120.
43. The complex of claim 42, wherein said gp120 is a part of an
HIV or SIV, is expressed on the surface of an HIV or SIV-infected cell
or is present in solution.
44. A complex comprising the fusion protein of claim 34 and HIV
or SIV gp120.
45. The complex of claim 44, wherein said gp120 is a part of an HIV or SIV, is
expressed on the
surface of an HIV or SIV infected cell or is present in solution.
46. A use of the fusion protein of claim 23 for
treating HIV or SIV infections in an animal.
47. The use of claim 46, wherein said animal is a human.

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48. A use of the fusion protein of claim 34 for
treating HIV or SIV infections in an animal.
49. The use of claim 48, wherein said animal is a human.
50. A method for the detection of HIV or SIV gp 120 in a sample, comprising
(a) contacting a sample suspected of containing HIV or SIV gp120 with the
fusion
protein of claim 23, wherein said fusion protein is detectably labeled, and
(b) detecting whether a complex is formed.
51. The method of claim 50, wherein said fusion protein is
detectably labeled.
52. A method for the detection of HIV or SIV gp120 in a sample,
comprising
(a) contacting a sample suspected of containing HIV or SIV
gp120 with the fusion protein of claim 34, and
(b) detecting whether a complex has formed.
53. The method of claim 52, wherein said fusion protein is
detectably labeled.
54. A use of the fusion protein of claim 23 for
the production of a medicament for treating HIV or SIV
infections in an animal.
55. A use of the fusion protein of claim 34 for
the production of a medicament for treating HIV or SIV
infections in an animal.

Description

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TITLE OF THE INVENTION
CLONED GENES ENCODING IG-CD4 FUSION PROTEINS AND THE USE THEREOF
F~ ELD OF THE INVENTION
The invention is in the field of recombinant genetics.
BACKGROUND OF THE INVENTION
The human and simian immunodeficiency viruses HIV and SIV are the
causative agents of Acquired Immune Deficiency Syndrome (AIDS) and
Simian Immunodef'iciency Syndrome (SIDS), respectively. See Curren, J.
stet al., cien ; x,:1359-1357 (1985); Weiss, R. et al., Nature
x:572-575 (1986). The HIV virus contains an envelope glycoprotein,
gp120 which binds to the CD4 protein present on the surface of helper
T lymphocytes, macrophages and other cells. Dalgleish et al. Nature,
x:763 (1984). After the gp120 binds to CD4, virus entry is facili-
tated by an envelope-mediated fusion of the viral target cell mem-
branes.
i

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During the course of infection, the host organism develops
antibodies against viral proteins, including the major envelope
glycoproteins c~p120 and gp4l. Despite this humoral immunity, the
disease progresses, resulting in a lethal immunosuppression charac-
terized by multiple opportunistic infections, parasitemia, dementia
and death. The failure of host anti-viral antibodies to arrest the
progression of the disease represents one of the most vexing and
alarming aspects of the infection, and augurs poorly for vaccination
efforts based upon conventional approaches.
Two factor's may play a role in the inefficacy of the humoral
response to immunodeficiency viruses. First, like other RNA viruses
(and like retroviruses in particular), the immunodeficiency viruses
show a high mutation rate which allows antigenic variation to progress
at a high rate in response to host immune surveillance. Second, the
envelope glycoproteins themselves are heavily glycosylated molecules
presenting few epitopes suitable for high affinity antibody binding.
The poorly antigenic, "moving" target which the viral envelope
presents, allows the host little opportunity for restricting viral
infection by specific antibody production.
Cells infected by the HIU virus express the gp120 glycoprotein on
their surface. Gp120 mediates fusion events among CD4+ cells via a
reaction similar to that by which the virus enters the uninfected
cell, leading to the formation of short-lived multinucleated giant
cells. Syncytium formation is dependent on a direct interaction of
the gp120 envelope glycoprotein with the CD4 protein. Dalgleish et
al., supra, Klatzmann, D. et al., Nature 312:763 (1984); McDougal,
J.S. et al. Science, 231:382 (1986); Sodroski, J. et al., Nature,
322:470 (1986); Lifson, J.D. et al., Nature, 323:725 (1986); Sodroski,
J. et al., Nature, 321:412 (1986).
The CD4 protein consists of a 370 amino acid extracellular region
containing four immunoglobulin-like domains, a membrane spanning
domain, and a charged intracellular region of 40 amino acid residues.

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Maddon, P. et al .. , Cel l 42:93 (1985) ; C1 ark, S. et al . , Proc. Natl .
Acad. Sci. yUSA~ 84:1649 (1987).
Evidence that CD4-gp120 binding is responsible for viral infec-
tion of cells bearing the CD4 antigen includes the finding that a
specific complex is formed between gp120 and CD4. McDougal et al.,
supra. Other workers have shown that cell lines, which were non-
infective for HIV, were converted to infectable cell lines following
transfection and expression of the human CD4 cDNA gene. Maddon et
al., Cell 47:33:3-348 (1986).
In contrast to the majority of antibody-envelope interactions,
the receptor-envelope interaction is characterized by a high affinity
(Ka = 1081/mole) immutable association. Moreover, the affinity of the
virus for CD4 is at least 3 orders of magnitude higher than the
affinity of CD4 for its putative endogenous ligand, the MHC class II
antigens. Indeed, to date, a specific physical association between
monomeric CD4 and class II antigens has not been demonstrated.
In response to bacterial or other particle infection, the host
organism usuallly produces serum antibodies that bind to specific
proteins or carbohydrates on the bacterial or particle surface,
coating the bacaeria. This antibody coat on the bacterium or other
particle stimulates cytolysis by Fc-receptor-bearing lymphoid cells by
antibody-dependent cellular toxicity (ADCC). Other serum proteins,
collectively called complement (C), bind to antibody-coated targets,
and also can coat foreign particles nonspecifically. They cause cell
death by lysis, or stimulate ingestion by binding to specific
receptors on the macrophage called complement receptors. See Darnell
J. et al., in Molecular Cell Biolo4y, Scientific American Books, pp.
641 and 1087 (1!386).
The most effective complement activating classes of human Ig are
IgM and IgGl. The complement system consists of 14 proteins that,
acting in order, cause lysis of cells. Nearly all of the C proteins
exist in normal serum as inactive precursors. When activated, some

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become highly specific proteolytic enzymes whose substrate is the next
protein in a sequential chain reaction.
The entire C sequence can be triggered by either of two initia-
tion pathways. In one (the classic pathway), Ab-Ag complexes bind and
activate C1, C4 and C2 to form a C3-splitting enzyme. In the second
pathway, polysaccharides commonly on the surface of many bacteria and
fungi bind with trace amounts of a C3 fragment and then with two other
proteins (factor B and properdin) to form another C3-splitting enzyme.
Once C3 is spl it by either pathway, the way is open for the remaining
sequence of steps which lead to cell lysis. See Davis, B.D., et al.,
In Microbiolo4v, 3rd ed., Harper and Row, Philadelphia, PA, pp. 452-
466 (1980).
A number of workers have disclosed methods for preparing hybrid
proteins. For example, Murphy, United States Patent 4,675,382 (1987),
discloses the use of recombinant DNA techniques to make hybrid protein
molecules by forming the desired fused gene coding for a hybrid
protein of diptheria toxin and a polypeptide ligand such as a hormone,
followed by exp~°ession of the fused gene.
Many workers have prepared monoclonal antibodies (Mabs) by
recombinant DNA techniques. Monoclonal antibodies are highly specific
well-characterized molecules in both primary and tertiary structure.
They have been widely used for in vitro immunochemical characteriza-
tion and quantitation of antigens. Genes for heavy and light chains
have been introduced into appropriate hosts and expressed, followed by
reaggregation of the individual chains into functional antibody
molecules (see, for example, Munro, Nature 312:597 (1984); Morrison,
S.L., Science X29:1202 (1985); Oi et al., Biotechniques 4:214 (1986);
Wood et al., Nature 314:446-449 (1985)). Light- and heavy-chain
variable regions have been cloned and expressed in foreign hosts
wherein they maintained their binding ability (Moore et al., European
Patent Application 0088994 (published September 21, 1983)).
Chimeric or hybrid antibodies have also been prepared by recom-
binant DNA techniques. Oi and Morrison, Biotechniques 4:214 (1986)

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describe a strategy for producing such chimeric antibodies which
include a chimeric human IgG anti-leu3 antibody.
Gascoigne, N.R.J., et al., Proc. Natl. Acad. Sci. (USA) 84:2936-
2940 (1987) disclose the preparation of a chimeric gene construct
containing a f-cell receptor a-chain variable (U) domain and the
constant (C) region coding sequence of an immunoglobulin .~2a molecule.
Cells transfected with the chimeric gene synthesize a protein product
that expresses immunoglobulin and T-cell receptor antigenic
determinants as well as protein A binding sites. This protein
associates with a normal a chain to form an apparently normal
tetrameric (H2L2, where H=heavy and L=light) immunoglobulin molecule
that is secreted.
Sharon, J., et al., Nature 309:54 (1984), disclose construct ion
of a chimeric gene encoding the variable (V) region of a mouse heavy
chain specific for the hapten azophenylarsonate and the constant (C)
region of a mouse kappa light chain (VHCK). This gene was introduced
into a mouse myeloma cell line. The chimeric gene was expressed to
give a protein which associated with light chains secreted from the
myeloma cell line to give an antibody molecule specific for
azophenylarsonate.
Morrison, :science 229:1202 (1985), discloses that variable light-
or variable heavy-chain regions can be attached to a non-Ig sequence
to create fusion proteins. This article states that the potential
uses for the fusion proteins are three: (1) to attach antibody
specifically to enzymes for use in assays; (2) to isolate non-Ig
proteins by ant igen columns; and (3) to specifically deliver toxic
agents.
Recent techniques for the stable introduction of immunoglobulin
genes into myeloma cells (Banerji, J., et al., Cell 33:729-740 (1983);
Potter, H., et al., Proc. Natl. Acad. Sci. I(USA) 81:7161-7165 (1984)),
coupled with detailed structural information, have permitted the use
of in vitro DNA methods such as mutagenesis, to generate recombinant
antibodies possessing novel properties.

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PCT Application W087/02671 discloses methods for producing
genetically engineered antibodies of desired variable region specifi-
city and constant region properties through gene cloning and expres-
sion of light and heavy chains. The mRNA from cloned hybridoma B cell
lines which praduce monoclonal antibodies of desired specificity is
isolated for cDNA cloning. The generation of light and heavy chain
coding sequences is accomplished by excising the cloned variable
regions and ligating them to light or heavy chain module vectors.
This gives cDNA sequences which code for immunoglobulin chains. The
lack of introns allows these cDNA sequences to be expressed in
prokaryotic hosts, such as bacteria, or in lower eukaryotic hosts,
such as yeast.
The generation of chimeric antibodies in which the antigen-
binding portion of the immunoglobulin is fused to other moieties has
been demonstrated. Examples of non-immunoglobulin genes fused to
antibodies include Staphylococcus aureus nuclease, the mouse oncogene
c-m~~c, and the Klenow fragment of . oli DNA polymerase I (Neuberger,
M.S., et al., Wa ur ~,:fi04-612 (1984); Neuberger, M.S., Trends in
biochemical Science, 347-349 (1985)).

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PCT Application W083/101533 describes chimeric antibodies wherein
the variable region of an in~nunoglobulin molecule is linked to a
portion of a second protein which may comprise the active portion of
an enzyme.
Boulianne ,et al., bature x;643 (1984) constructed an immuno-
globulin gene in which the DNA segments that encode mouse variable
regions specific for the hapten trinitrophenol (TNP) are joined to
segments that encode human mu and kappa regions. These chimeric genes
were expressed t:o give functional TNP-binding chimeric IgM.
Morrison et al., P.N.A.S. (USA) x:6851 (1984), disclose a
chimeric molecule utilizing the heavy-chain variable region exons of
an anti-phosphoryl choline myeloma protein G, which were joined to the
exons of either human kappa light-chain gene. The genes were trans-
fected into mouse myeloma cell lines, generating transformed cells
that produced chimeric mouse-human IgG with antigen-binding function.
Despite the progress that has been achieved on determining the
mechanism of HIV infection, a need continues to exist for methods of
treating HIV viral infections.
SUMMARY OF THE INVENTION
The i nvent i on rel ates to a gene compri s i ng a DNA sequence wh i ch
encodes a fusion protein comprising 1) CD4, or a fragment thereof
which binds to HIV gp120, and 2) an immunoglobulin light or heavy
chain; wherein said CD4 or HIV gp120-binding fragment thereof replaces
the variable region of the light or heavy immunoglobulin chain.
The inventian also relates to vectors containing the gene of the
invention and hosts transformed with the vectors.
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1340'41
The invent:ion also relates to a method of producing a fusion
protein comprising CD4, or fragment thereof which binds to HIV gp120,
and an immunoglobulin light or heavy chain, wherein the variable
region of the immunoglobulin light or heavy chain has been substituted
with CD4, or HIV gp120-binding fragment thereof, which comprises:
cultivating in a nutrient medium under protein produc-
ing conditions, a host strain transformed with the vector
containing the gene of the invention, said vector further
comprising expression signals which are recognized by said host
strain and direct expression of said fusion protein, and
recovering the fusion protein so produced.
The invention also relates to a fusion protein comprising CD4, or
fragment thereof which is capable of binding to HIV gp120, fused at
the C-terminus to a second protein which comprises an immunoglobulin
light or heavy chain, wherein the variable region of said light or
heavy chain is substituted with CD4 or a HIV gp120 binding fragment
thereof.
The invention also relates to an immunoglobulin-like molecule
comprising the fusion protein of the invention together with an
immunoglobulin light or heavy-chain, wherein said immunoglobulin like
molecule binds HIV gp120.
The IgGI fusion proteins and immunoglobulin-like molecules may be
useful for both complement-mediated and cell-mediated (ADCC) immunity,
while the IgM fusion proteins are useful principally through comple-
ment-mediated immunity.
The invention also relates to a complex between the fusion
proteins and immunoglobulin-like molecule of the invention and HIV
gp120.
The invention also relates to a method for treating HIV or SIV
infections comprising administering the fusion protein or immunoglobu-
lin-like molecule of the invention to an animal.
The invention further relates to a method for detecting HIV gp120
in a sample comprising contacting a sample suspected of containing HIV

~34o~4i
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or gp120 with t he fusion protein or immunoglobulin-like molecule of
the invention, and detecting whether a complex has formed.
QESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention is directed to a protein gene which comprises
1 ) a DNA sequence whi ch codes for CD4, or fragment thereof
which binds to HIV gp120, fused to
2) a DNA sequence which encodes an immunoglobulin heavy
chain.
Preferably., the antibody has effector function.
The invention is also directed to a protein gene which comprises
1) a DNA sequence which codes for CD4, or fragment thereof
which binds to HIV gp120, fused to
2) a DNA sequence which encodes an immunoglobulin light
chain; wherein said sequence which codes for CD4, or HIV
gp120~-binding fragment thereof, replaces the variable region
of the light immunoglobulin chain.
The i nventi on i s al so di rected to the express i on of these novel
fusion proteins in transformed hosts and the use thereof to treat and
diagnose HIV infections. In particular, the invention relates to
expressing said genes in mammalian hosts which express complementary
light or heavy chain immunoglobulins to give immunoglobulin-like
molecules which have antibody effector function and also bind to HIV
or SIV gp120.
The term "antibody effector function" as used herein denotes the
ability to fix complement or to activate ADCC.
The fusion proteins and immunoglobulin-like molecules may be
administered to~ an animal for the purpose of treating HIV or SIV
infections. By the terms "HIV infections" is intended the condition
of having AIDS, AIDS related complex (ARC) or where an animal harbors
the AIDS virus, but does not exhibit the clinical symptoms of AIDS or

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ARC. By the terms "SIN infections" is intended the condition of being
infected with simian immunodeficiency virus.
By the term "animal" is intended all animals which may derive
benefit from the administration of the fusion proteins and immuno-
globulin-like molecules of the invention. Foremost among such animals
are humans, however, the invention is not intended to be so limited.
By the term "fusion protein" is intended a fused protein
comprising CD4,, or fragment thereof which is capable of binding to
gp120, linked at its C-terminus to an immunoglobulin chain wherein a
portion of the N-terminus of the immunoglobulin is replaced with CD4.
In general, that portion of immunoglobulin which is deleted is the
variable region. The fusion proteins of the invention may also
comprise immunoglobulins where more than just the variable region has
been deleted and replaced with CD4 or HIY gp120 binding fragment
thereof. For example, the UH and CH1 regions of an immunoglobulin
chain may be deleted. Preferably, any amount of the N-terminus of the
immunoglobulin heavy chain can be deleted as long as the remaining
fragment has antibody effector function. The minimum sequence
required for binding complement encompasses domains CH2 and CH3.
Joining of Fc portions by the hinge region is advantageous for
increasing the efficiency of complement binding.
The CD4 portion of the fusion protein may comprise the complete
CD4 sequence, the 370 amino acid extracellular region and the membrane
spanning domain, or the extracellular region. The fusion protein may
comprise fragments of the extracellular region obtained by cutting the
DNA sequence which encodes CD4 at the BspMl site at position 514 or
the PvuII sites at position 629 (see Table 1) to give nucleotide
sequences which encode CD4 fragments which retain binding to gp120.
In general, any fragment of CD4 may be used as long as it retains
binding to gp12~0.
Where the fusion protein comprises an immunoglobulin light chain,
it is necessary that no more of the Ig chain be deleted than is
necessary to form a stable complex with a heavy chain Ig. In particu-

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lar, the cysteine residues necessary for disulfide bond formation must
be preserved on both the heavy and light chain moieties.
When expressed in a host, e.g., a mammalian cell, the fusion
protein may associate with other light or heavy Ig chains secreted by
the cell to giwe a functioning itntnunoglobulin-like molecule which is
capable of binding to gp120. The gp120 may be in solution, expressed
on the surface of infected cells, or may be present on the surface of
the HIV virus itself. Alternatively, the fusion protein may be
expressed in a mammalian cell which does not secrete other light or
heavy Ig chains.. When expressed under these conditions, the fusion
protein may form a homodimer.
Genomic or CDNA sequences may be used in the practice of the
invention. Genomic sequences are expressed efficiently in myeloma
cells, since they contain native promoter structures.
The constant regions of the antibody cloned and used in the
chimeric immunoglobulin-like molecule may be derived from any mam-
malian source. The constant regions may be complement binding or ADCC
active. However, preliminary work (see Examples) indicates that the
fusion proteins of the invention may mediate HIV or SIV infected cell
death by an ADCC or complement-independent mechanism. The constant
regions may be derived from any appropriate isotype, including IgGl,
IgG3, or IgM.
The joining of various DNA fragments, is performed in accordance
with conventional techniques, employing blunt-ended or staggered-ended
termini for liqation, restriction enzyme digestion to provide appro-
priate termini, filling in of cohesive ends as appropriate, alkali and
phosphatase treatment to avoid undesirable joining, and ligation with
appropriate ligases. The genetic construct may optionally encode a
leader sequence to allow efficient expression of the fusion protein.
For example, the leader sequence utilized by Maddon et al., Cell
42:93-104 (1985) for the expression of CD4 may be used.
For cDNA, the cDNA may be cloned and the resulting clone screen-
ed, for example, by use of a complementary probe or by assay for

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expressed CD4 using an antibody as disclosed by Dalgleish et al.,
Na a a 3~?:763-766 (1984); Klatzmann et al., Immunol. Today 7:291-297
(1986); McDougal et al., J. Immunol. 135:3151-3162 (1985); and
McDougal, J. et al., J. Immunol. 137:2937-2944 (1986).
To express the fusion hybrid protein, transcriptional and
translational signals recognized by an appropriate host element are
necessary. Eukaryotic hosts which may be used include mammalian cells
capable of culture in vitro, particularly leukocytes, more particu-
larly myeloma cells or other transformed or oncogenic lymphocytes,
e.g., EBU-transformed cells. Alternatively, non-mammalian cells may
be employed, such as bacteria, fungi, e.g., yeast, filamentous fungi,
or the like.
Preferred hosts for fusion protein production are mammalian
cells, grown in vitro in tissue culture or in vivo in animals.
Mammalian cells provide post translational modification to immuno-
globulin protein molecules which provide for correct folding and
glycosylation of appropriate sites. Mammalian cells which may be
useful as hosts include cells of fibroblast origins such as UERO or
CHO-K1 or cells of lymphoid origin, such as the hybridoma SP2/0-AG14
or the myeloma P3x63Sgh, and their derivatives. For the purpose of
preparing an immunoglobulin-like molecule, a plasmid containing a gene
which encodes a heavy chain immunoglobulin, wherein the variable
region has been replaced with CD4 or fragment thereof which binds to
gp120, may be introduced, for example, into J558L myeloma cells, a
mouse plasmacyt,oma expressing the lambda-1 light chain but which does
not express a heavy chain (see Oi et al., P.N.A.S. (USA) 80:825-829
(1983)). Other preferred hosts include COS cells, BHK cells and
hepatoma cells.
The constructs may be joined together to form a single DNA
segment or may be maintained as separate segments, by themselves or in
conjunction with vectors.

-I3- 1340'~4~.
Where the fusion protein is not glycosylated, any host may be
used to express the protein which is compatible with replicon and
control sequences in the expression plasmid. In general, vectors
containing replicon and control sequences are derived from species
compatible with a host cell are used in connection with the host. The
vector ordinarily carries a replicon site, as well as specific genes
which are capable of providing phenotypic selection in transformed
cells. The expression of the fusion protein can also be placed under
control with other regulatory sequences which may be homologous to the
organism in its untransformed state. For example, lactose-dependent
E. coli chromasomal DNA comprises a lactose or lac operon which
mediates lactose utilization by elaborating the enzyme beta-galacto-
sidase. The lac control elements may be obtained from bacterial phage
lambda plac5, which is infective for E. coli. The lac promoter-
operator system can be induced by IPTG.
Other promoters/operator systems or portions thereof can be
employed as well. For example, colicin E1, galactose, alkaline
phosphatase, tryptophan, xylose, tax, and the like can be used.
For mammalian hosts, several possible vector systems are avail-
able for expression. One class of vectors utilize DNA elements which
are derived from animal viruses such as bovine papilloma virus,
polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses
(RSV, MMTV or MOMLV), or SV40 virus. Cells which have stably inte-
grated the DNA i nto thei r chromosomes may be sel ected by i ntroduc i ng
one or more markers whi ch al l ow sel ecti on of transfected host cel 1 s .
The marker may provide for prototropy to an auxotrophic host, biocide
resistance, e.g., antibiotics, or heavy metals such as copper or the
like. The selectable marker gene can be either directly linked to the
DNA sequences to be expressed, or introduced into the same cell by
cotransformation. Additional elements may also be needed for optimal
synthesis of mRNA. These elements may include splice signals, as well
as transcriptional promoters, enhancers, and termination signals. The

1340'~~~.
-14-
cDNA expression vectors incorporating such elements includes those
described by Ok,ayama, H., Mol. Cel. Biol., 3:280 (1983) and others.
Once the vector or DNA sequence containing the constructs has
been prepared for expression, the DNA constructs may be introduced to
an appropriate host. Various techniques may be employed, such as
protoplast fusion, calcium phosphate precipitation, electroporation or
other conventional techniques. After the fusion, the cells are grown
in media and screened for the appropriate activity. Expression of the
genes) results in production of the fusion protein. This expressed
fusion protein may then be subject to further assembly to form the
immunoglobulin-like malecule.
The host cells for immunoglobulin production may be immortalized
cells, primarily myeloma or lymphoma cells. These cells may be grown
in appropriate nutrient medium in culture flasks or injected into a
synergistic host, e.g., mouse or a rat, or immunodeficient host or
host site, e.g., nude mouse or hamster pouch. In particular, the
cells may be introduced into the abdominal cavity of an animal to
allow production of ascites fluid which contains the immunoglobulin-
like molecule. Alternatively, the cells may be injected subcutane-
ously.and the chimeric antibody is harvested from the blood of the
host. The cells may be used in the same manner as hybridoma cells.
See Diamond et, al., N. Enq. J. Med. 304:1344 (1981), and Kennatt,
McKearn and Bechtol (Eds.), Monoclonal Antibodies: Hvbridomas: -- A
New Dimension in Biolo4ic Analysis, Plenum, 1980.
The fusion proteins and immunoglobulin-like molecules of the
invention may be isolated and purified in accordance with conventional
conditions, such as extraction, precipitation, chromatography,
affinity chromatography, electrophoresis or the like. For example,
the IgGl fusion proteins may be purified by passing a solution through
a column which contains immobilized protein A or protein G which
selectively binds the Fc portion of the fusion protein. See, for
example, Reis, K.J., et al., J. Immunol. 132:3098-3102 (1984); PCT
Application, Publication No. W087/00329. The chimeric antibody may

-15- 1~ ~p7 4i
the be eluted by treatment with a chaotropic salt or by elution with
aqueous acetic acid (1 M_).
Alternatively the fusion proteins may be purified on anti-CD4
antibody columns, or on anti-immunoglobulin antibody columns.
In one embodiment of the invention, cDNA sequences which encode
CD4, or a fragment thereof which binds gp120, may be ligated into an
expression plasmid which codes for an antibody wherein the variable
region of the gene has been deleted. Methods for the preparation of
genes which encode the heavy or light chain constant regions of
immunoglobulins are taught, for example, by Robinson, R. et al., PCT
Application, Publication No. W087-02671.
Preferred immunoglobulin-like molecules which contain CD4, or
fragments thereof, contain the constant region of an IgM, IgGl or IgG3
antibody which binds complement at the Fc region.
The fusion protein and immunoglobulin-like molecules of the
invention may be used for the treatment of HIV viral infections. The
fusion protein complexes to gp120 which is expressed on infected
cells. Although the inventor is not bound by a particular theory, it
appears that the Fc portion of the hybrid fusion protein may bind with
complement, which mediates destruction of the cell. In this manner,
infected cells are destroyed so that additional viral particle
production is atopped.
For the purpose of treating HIV infections, the fusion protein or
immunoglobulin~-like molecule of the invention may additionally contain
a radiolabel or therapeutic agent which enhances destruction of the
HIV particle or HIV-infected cell.
Examples of radioisotopes which can be bound to the fusion
protein or immunoglobulin-like molecule of the invention for use in
HIV-therapy are 125I, 131I~ 90Y~ 67Cu~ 217gi~ 211At~ 212pb~ 47Sc, and
109pd. Optionally, a label such as boron can be used which emits a
and ~ particles upon bombardment with neutron radiation.

-16-
1~~~'~4~
For in vivo diagnosis radionucleotides may be bound to the fusion
protein or immunoglobulin-like molecule of the invention either
directly or by using an intermediary functional group. An inter-
mediary group which is often used to bind radioisotopes, which exist
as metallic c.ations, to antibodies is diethylenetriaminepentaacetic
acid (DTPA). Typical examples of metallic cations which are bound in
this manner are 99mTc 123I, 111In~ 131I~ 9~Ru, 6~Cu, 6~Ga, and 68Ga.
Moreover, the fusion protein and immunoglobulin-like molecule of
the invention may be tagged with an NMR imaging agent which include
paramagnetic atoms. The use of an NMR imaging agent allows the in
vivo diagnosis of the presence of and the extent of HIU infection
within a patient using NMR techniques. Elements which are particu-
larly useful in this manner are IS~Gd, 55Mn, 162Dy, 52Cr, and 56Fe.
Therapeutic agents may include, for example, bacterial toxins
such as diphtheria toxin, or ricin. Methods for producing fusion
proteins comprising fragment A of diphtheria toxin are taught in U.S.
Patent 4,675,382 (1987). Diphtheria toxin contains two polypeptide
chains. The B chain binds the toxin to a receptor on a cell surface.
The A chain actually enters the cytoplasm and inhibits protein
synthesis by inactivating elongation factor 2, the factor that
translocates ribosomes along mRNA concomitant with hydrolysis of ETP.
See Darnell, J., et al., in Molecular Cell Biolo4y, Scientific
American Books, Inc., page 662 (1986). Alternatively, a fusion
protein comprising ricin, a toxic lectin, may be prepared.
Introduction of the chimeric molecules by gene therapy may also
be contemplated, for example, using retroviruses or other means to
introduce the genetic material encoding the fusion proteins into
suitable target tissues. In this embodiment, the target tissues
having the cloned genes of the invention may then produce the fusion
protein in viva.
The dose ranges for the administration of the fusion protein or
immunoglobulin-like molecule of the invention are those which are

-1~- 1340'41
large enough to produce the desired effect whereby the symptoms of HIV
or SIV infection are ameliorated. The dosage should not be so large
as to cause adverse side effects, such as unwanted cross-reactions,
anaphylactic reactions, and the like. Generally, the dosage will vary
with the age, condition, sex and extent of disease in the patient,
counterindications, if any, i~nune tolerance and other such variables,
to be adjusted by the individual physician. Dosage can vary from .O1
mg/kg to 50 mg/kg, preferably 0.1 mg/kg to 1.0 mg/kg, of the immuno-
globulin-like molecule in one or more administrations daily, for one
or several days. The immunoglobulin-like molecule can be administered
parenterally b;y injection or by gradual perfusion over time. They can
be administered intravenously, intraperitoneally, intramuscularly, or
subcutaneously.
Preparations for parenteral administration include sterile or
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water, alco-
holic/aqueous solutions, emulsions or suspensions, including saline
and buffered media. Parenteral vehicles include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's, or fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers, such as those based
on Ringer's dextrose, and the like. Preservatives and other additives
may also be present, such as, for example, antimicrobials, anti-
oxidants, chelating agents, inert gases and the like. See, generally,
Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.
The invention also relates to a method for preparing a medicament
or pharmaceutical composition comprising the components of the
invention, the medicament being used for therapy of HIV or SIV
infection in animals.
The detection and quantitation of antigenic substances and
biological samples frequently utilized immunoassay techniques. These

-18- 134741
techniques are based upon the formation of the complex between the
antigenic substance, e.g., gp120, being assayed and an antibody or
antibodies in which one or the other member of the complex may be
detectably labeled. In the present invention, the immunoglobulin-like
molecule or fusion protein may be labeled with any conventional label.
Thus, the hybrid fusion protein or immunoglobulin-like molecule
of the invention can also be used in assay for HIV or SIV viral
infection in a biological sample by contacting a sample, derived from
an animal suspected of having an HIV or SIV infection, with the fusion
protein or immunoglobulin-like molecule of the invention, and
detecting whether a complex with gp120, either alone or on the surface
of an HIV-infected cell, has formed.
For example, a biological sample may be treated with nitro-
cellulose, or other solid support which is capable of immobilizing
cells, cell particles or soluble protein. The support may then be
washed with suitable buffers followed by treatment with the fusion
protein which may be detectably labeled. The solid phase support may
then be washed with the buffer a second time to remove unbound fusion
protein and the label on the fusion protein detected.
In carrying out the assay of the present invention on a sample
containing gp120, the process comprises:
a) contacting a sample suspected containing gp120 with a
solid support to effect immobilization of gp120, or cell which
expresses gp120 on its surface;
b) contacting said solid support with the detectably labeled
immunoglobulin--like molecule or fusion protein of the invention;
c) incubating said detectably labeled immunoglobulin-like
molecule with said support for a sufficient amount of time to allow
the immunoglobulin-like molecule or fusion protein to bind to the
immobilized gpJ'.20 or cell which expresses gp120 on its surface;
d) separating the solid phase support from the incubation
mixture obtained in step c); and

-19- 1340741
e) detecting the bound immunoglobulin-like molecule or
fusion protein and thereby detecting and quantifying gp120.
Alternatively, labeled immunoglobulin-like molecule (or fusion
protein) -gp120 complex in a sample may be separated from a reaction
mixture by cantacting the complex with an immobilized antibody or
protein which is specific for an immunoglobulin or, e.g., protein A,
protein G, anti-IgM or anti-IgG antibodies. Such anti-immunoglobulin
antibodies may be monoclonal or polyclonal. The solid support may
then be washed with suitable buffers to give an immobilized gp120-
labeled immunaglobulin-like molecule antibody complex. The label on
the fusion protein may then be detected to give a measure of
endogenous gp120 and, thereby, the presence of HIV.
This aspect of the invention relates to a method for detecting
HIV or SIV viral infection in a sample comprising
(a) contacting a sample suspected of containing gp120 with
a fusion protein or immunoglobulin-like molecule
comprising CD4, or fragment thereof which binds to
gp120, and the Fc portion of an immunoglobulin chain,
(b) detecting whether a complex is formed.
The invention also relates to a method of detecting gp120 in a
sample, further comprising
(c) contacting the mixture obtained in step (a) with an Fc
binding molecule, such as an antibody, protein A, or
protein G, which is immobilized on a solid phase
support and is specific for the hybrid fusion protein,
to give a gp120 fusion protein-immobilized antibody
complex
(d) washing the solid phase support obtained in step (c) to
remove unbound fusion protein,
(e) and detecting the label on the hybrid fusion protein.
Of course, the specific concentrations of detectably labeled
immunoglobulin-like molecule (or fusion protein) and gp120, the
temperature and time of incubation, as well as other assay conditions

_20- 13 4 0'~ 41
may be varied, depending on various factors including the concentra-
tion of gp120 in the sample, the nature of the sample, and the like.
Those skilled in the art will be able to determine operative and
optimal assay conditions for each determination by employing routine
experimentation.
Other such steps as washing, stirring, shaking, filtering and the
like may be added to the assays as is customary or necessary for the
particular situation.
One of the ways in which the immunoglobulin-like molecule or
fusion protein of the present invention can be detectably labeled is
by linking the same to an enzyme. This enzyme, in turn, when later
exposed to its substrate, will react with the substrate in such a
manner as to produce a chemical moiety which can be detected as, for
example, by spectrophotometric, fluorometric or by visual means.
Enzymes which c an be used to detectably label the immunoglobulin-like
mol ecul a or fus i on protei n of the present i nvent i on i ncl ude, but are
not limited to, malate dehydrogenase, staphylococcal nuclease, delta-
11-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophos-
phate dehydrogenase, triose phosphate isomerase, horseradish
peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-
galactosidase, ribonuclease, urease, catalase, glucose-UI-phosphate
dehydrogenase, glucoamylase and acetylcholine esterase.
The immunoglobulin-like molecule or fusion protein of the present
invention may also be labeled with a radioactive isotope which can be
determined by such means as the use of a gamma counter or a
scintillation counter or by autoradiography. Isotopes which are
particularly useful for the purpose of the present invention are: 3H,
125I~ 131I~ 32p~ 355 14C~ 5lCr~ 36C1~ 57Co~ 58Co, 59Fe and 75Se.
It is also possible to label the immunoglobulin-like molecule or
fusion protein with a fluorescent compound. When the fluorescently
labeled immunoglobulin-like molecule is exposed to light of the proper
wave length, it,s presence can then be detected due to the fluorescence

m~a~~~
-21-
of the dye. Among the most commonly used fluorescent labelling
compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin,
phycocyanin, a'llophycocyanin, o_-phthaldehyde and fluorescamine.
The immunoglobulin-like molecule or fusion protein of the
invention can also be detectably labeled using fluorescence emitting
metals such as 152Eu, or others of the lanthanide series. These
metals can be attached to the immunoglobulin-like molecule or fusion
protein using such metal chelating groups as diethylenetriaminepenta-
acetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The immunoglobulin-like molecule or fusion protein of the present
invention also can be detectably labeled by coupling it to a
chemiluminescent compound. The presence of the chemiluminescent-
tagged immunoglobulin-like molecule or fusion protein is then
determined by detecting the presence of luminescence that arises
during the course of a chemical reaction. Examples of particularly
useful chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester.
Likewise, a bioluminescent compound may be used to label the
immunoglobulin-like molecule or fusion protein of the present
invention. Bioluminescence is a type of chemiluminescence found in
biological systems in which a catalytic protein increases the
efficiency of the chemiluminescent reaction. The presence of a
bioluminescent protein is determined by detecting the presence of
luminescence. Important bioluminescent compounds for purposes of
labeling are luciferin, luciferase and aequorin.
Detection of the immunoglobulin-like molecule or fusion protein
may be accomplished by a scintillation counter, for example, if the
detectable label is a radioactive gamma emitter, or by a fluorometer,
for example, if the label is a fluorescent material. In the case of
an enzyme label, the detection can be accomplished by colorimetric
methods which employ a substrate for the enzyme. Detection may also
be accomplished by visual comparison of the extent of enzymatic

~~4p~ 41
-22-
reaction of a substrate in comparison with similarly prepared
standards.
The assay of the present invention is ideally suited for the
preparation of a kit. Such a kit may comprise a carrier means being
compartmentalized to receive in close confinement therewith one or
more container means such as vials, tubes and the like, each of said
container means comprising the separate elements of the immunoassay.
For example, there may be a container means containing a solid phase
support, and further container means containing the detectably labeled
immunoglobulin-like molecule or fusion protein in solution. Further
container means may contain standard solutions comprising serial
dilutions of analytes such as gp120 or fragments thereof to be
detected. The standard solutions of these analytes may be used to
prepare a standard curve with the concentration of gp120 plotted on
the abscissa and the detection signal on the ordinate. The results
obtained from a sample containing gp120 may be interpolated from such
a plot to give the concentration of gp120.
The immunoglobulin-like molecule or fusion protein of the present
invention can also be used as a stain for tissue sections. For
example, a labeled immunoglobulin-like molecule comprising CD4 or
fragment thereof which binds to gp120 may be contacted with a tissue
section, e.g., a brain biopsy specimen. This section may then be
washed and the label detected.
The following examples are illustrative, but not limiting the
method and composition of the present invention. Other suitable
modifications and adaptations which are obvious to this skill in the
art are within the spirit and scope of this invention.
EXAMPLES
Example 1: Preparation of CD4-I9 cDNA Constructs

. 1340'41
-23-
The extracellular portion of the CD4 molecule (See Madden, P.J.,
etet al., ~1 4_x:93-104 (1985)) was fused at three locations in a human
IgGl heavy chain constant region gene by means of a synthetic splice
donor linker molecule. To exploit the splice donor linker, a BamHI
linker having the sequence CGCGGATCCGCG was first inserted at amino
acid residue :395 of the CD4 precursor sequence (nucleotide residue
1295). A synthetic splice donor sequence
GATCCCGAGGGTGAGTACTA
GGCTCCCACTCATGATTCGA
bounded by BamHI and HindIII complementary ends was created and fused
to the HindIIl: site in the intron preceding the CH1 domain, to the
EspI site in i;he intron preceding the hinge domain, and to the BanI
site preceding the CH2 domain of the IgGl genomic sequence. Assembly
of the chimeric: genes by ligation at the BamHI site afforded molecules
in which either the variable (V) region, the V+CH1 regions, or the V,
CH1 and hinge regions were replaced by CD4. In the last case, the
chimeric molecule is expected to form a monomer structure, while in
the former, a dimeric molecule is expected.
On such genetic construct which contains the DNA sequence which
encodes CD4 linked to human IgGl at the Hind3 site upstream of the CH1
region (fusion protein CD4H~1) is depicted in Table 1. The plasmid
containing this genetic construct (pCD4H~1) has been deposited in E.
coli {MC1061/P;3) at the American Type Culture Collection (ATCC) under
the terms of the Budapest Treaty and given accession number 67611.
A second genetic construct which contains the DNA sequence which
encodes CD4 linked to human IgGl at the Esp site upstream of the hinge
region (fusion protein CD4ET1) is depicted in Table 2. The plasmid
containing this genetic construct (pCD4E~1) has been deposited in E.
coli (MC1061/P3) at the ATCC under the terms of the Budapest Treaty
and given accession number 67610.
A third genetic construct which contains the DNA sequence which
encodes CD4 linked to human IgM at the Mst2 site upstream of the CHl
region (fusion protein CD4M~) is depicted in Table 3. The plasmid

. ,.
..~ 1340'41
-24-
containing this genetic construct (pCD4MU) has been deposited in E.
(MC1061/P3) at the ATCC under the terms of the Budapest Treaty
and given accession number 67609.
A fourth genetic construct which contains the DNA sequence which
encodes CD4 1 i nked to human IgM at the Pst si to upstream of the CH2
region (fusion protein CD4PU) is depicted in Table 4. The plasmid
containing this genetic construct (pCD4PU) has been deposited in E.
coli (MC1061/P3) at the ATCC under the terms of the Budapest Treaty
and given accession number 67608.
A fifth genetic construct which contains the DNA sequence which
encodes CD4 linked to human IgGl at the and site downstream from the
hinge region (fusion protein CD4B~1) is depicted in Table 5.
Two simillar constructs were prepared from the human IgM heavy
chain constant region by fusion with the introns upstream of the ~ CH1
and CH2 domains at an MstII site and a PstI site respectively. The
fusions were made by joining the PstI site of the CD4/IgGI construct
fused at the E:sp site in IgGl gene to the MstII and Pst sites in the
IgM gene. In the first instance, this was performed by treatment of
the Pst end with T4 DNA Polymerase and the MstII end with . coli DNA
Polymerase, followed by ligation; and in the second instance, by
ligation alone.
Immunoprecipitation of the fusion proteins with a panel of
monoclonal antibodies directed against CD4 epitopes showed that all of
the epitopes were preserved. A specific high affinity association is
demonstrated between the chimeric molecules and HIV envelope proteins
expressed on the surface of cells transfected with an attenuated
(reverse transc:riptase deleted) proviral construct.
~i

_ -25-
Tabl a 1 13 4 0'~ 4 ~.
FN S B
N S B M H DHA S
U P B N G RAU T
4 B V L A AE9 X
H 2 1 1 1 236 1
GCCTGTTTGAGAAGCAGCGGGCAAGAAAGACGCAAGCCCAGAGGCCCTGCCATTTCTGTG
1 _________.,_________,_________,_________,___ _,_________, 60
CGGACAAA(:TCTTCGTCGCCCGTTCTTTCTGCGTTCGGGTCTCCGGGACGGTAAAGACAC
B PS S S
DBS ADNPA D DHNA M HM HNC
DAP VRL.W D RALU N AN PCR
EN1 AAA,M9 E AEA9 L EL AIF
122 22416 1 2346 1 31 211
GGCTCAGGTCCCTACTGGCTCAGGCCCCTGCCTCCCTCGGCAAGGCCACAATGAACCGGG
61 ---______;_________,_________,_________,__ _,_________, 120
CCGAGTCCAGGGATGACCGAGTCCGGGGACGGAGGGACCCGTTCCGGTGTTACTTGGCCC
M N R G -
H F F
I B N HH N M D
N B U HA U N D
F V 4 AE 4 L E
1 1 H 12 H 1 1
GAGTCCCTT'TTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGCAGCCACTC
121 -________.,_________,_________*_________,_________+_________, 180
CTCAGGGAAAATCCGTGAACGAAGACCACGACCTTGACCGCGAGGAGGGTCGTCGGTGAG
V P F R H L L L V L Q L A L L P A A T Q-
B E E R A
B C C S L
V 0 0 A U
1 K K 1 1
AGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGCTT
181 _________*_________+_________,_________,__ _,_________, 240
TCCCTTTCTTTCACCACGACCCGTTTTTTCCCCTATGTCACCTTGACTGGACATGTCGAA
G K K V V L G K K G D T V E L T C T A S-

-26- 1340?41,
H
M M I
B B N
0 0 F
2 2 1
CCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAA
241 _________,_________,_________,_________,_________,__ _~ 300
GGGTCTTC'TTCTCGTATGTTAAGGTGACCTTTTfGAGGTTGGTCTATTTCTAAGACCCTT
Q K K S I Q F H W K N S N Q I K I L G N -
B S S F H
NBS F AA A A N H I
LAP 0 VU L U U H N
AN1 K A9 U 3 D A F
422 1 26 1 A 2 1 1
ATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAA
301 ________..,_________,_________,_________,_________,_________, 360
TAGTCCCGAGGAAGAATTGATTTCCAGGTAGGTTCGACTTACTAGCGCGACTGAGTTCTT
Q G :> F L T K G P S K L N D R A D S R R _
S S H H
MANAS BA I A I D
BVLUT CU N F N D
0AA9Y L3 F L F E
22461 lA 1 2 1 1
GAAGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACT
3g1 _________,_________,_________,___._____,.________,_________, 420
CTTCGGAAACCCTGGTTCCTTTGAAGGGGGACTAGTAGTTCTTAGAATTCTATCTTCTGA
S L W D Q G N F P L I I K N L K I E D S-
S
M M AMAM M
B N VNUN A
0 L AL9L E
2 1 2161 1
CAGATACTT,ACATCTGTGAAGTGGAGGACCACAAGGACGAGGTGCAATTGCTAGTGTTCG
421 _________,_________,_________,_________,_________,_________, 480
GTCTATGAA'tGTAGACACTTCACCTCCTGGTCTTCCTCCTCCACGTTAACGATCACAAGC
D T Y I C E V E D Q K E E V Q L L V F G_

n
- 1340'41
B
S S
P T
M Y
1 1
GATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCACAGCCTGACCCTGACCTTGG
481 -________,_________,_________,_________,_________,_________, 540
CTAACTGACGGTTGAGACTGTGGGTGGACGAACTCCCCGTCTCGGACTGGGACTGGAACC
L T A N S D T H L L Q G Q S L T L T L E-
8 BS H
BS SC D M I S
AP TR D N N T
N1 NF E L F Y
22 11 1 1 1 1
/ /
AGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATAC
541 -________,_________,_________,_________,_________+_________, 600
TCTCGGGGGGACCATCATCGGGGAGTCACGTTACATCCTCAGGTTCCCCATTTTTGTATG
S P P G S S P S V Q C R S P R G K N I Q -
N BBH S B BS
M MD ASP A BSSGSC S B N SC
B ND LPV L APTIAR T A L TR
0 LE UBU U NiNACF X N A NF
2 11 122 1 221111 1 1 4 11
AGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACAT
601 _________,..________,_________,_________,_________,_________, 660
TCCCCCCCTTCTGGGAGAGGCACAGAGTCGACCTCGAGGTCCTATCACCGTGGACCTGTA
G G K T L S V S Q L E L Q D S G T W T C-
N
NS M NM A
LP B HA L
AH 0 EE U
31 2 11 1
GCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTT
661 -________,_________,_________,_________,_________,_________, 720
CGTGACAGAACGTCTTGGTCTTCTTCCACCTCAAGTTTTATCTGTAGCACCACGATCGAA
T V L Q N Q K K V E F K I D I V V L A F-

r-.
-28- 13 4 0'~ 4 ~.
HS M M
AT N N
EU L L
31 1 1
TCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCC
721 -________,_________~_________t_________,_________+_________, 780
AGGTCTTCCGGAGGTCGTATCAGATATTCTTTCTCCCCCTTGTCCACCTCAAGAGGAAGG
Q K A S S I V Y K K E G E Q V E F S F P-
A A M
L L N
U U L
1 1 i
CACTCGCCTTTACAGTTGAAAAGCTGACGGCCAGTGGCGAGCTGTCGTCGCAGGCGGAGA
781 _________r_________~_________~_________t_________~_________
~ 840
GTGAGCGGAA,ATGTCAACTTTTCGACTGCCCGTCACCGCTCGACACCACCGTCCGCCTCT
L A F T V E K L T G S G E L W W Q A E R-
P S
H M FM A M
P N LN U B
H L ML 3 0
1 1 11 A 2
GGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAAGTGTCTGTAA
841 -________,_________+_________,_________,_________~_________, 900
CCCGAAGGAGGAGGTTCAGAACCTAGTGGAAACTGGACTTCTTGTTCCTTCACAGACATT
A S S S K S W I T F D L K N K E V S V K-
B BS PS
SM SCADNPAD A A H
TA TRVRLUUD L L P
EE NFAAAM9E U U H
23 11224161 1 1 1
/ / / //
AACGGGTTACCCAGGACCCTAAGCTCCAGATGGCCAAGAAGCTCCCGCTCCACCTCACCC
901 -________~_________,_________~_________,_________,_________, 960
TTGCCCAATGGGTCCTGGGATTCGAGGTCTACCCGTTCTTCGAGGGCGAGGTGGAGTGGG
R V T Q D P K L Q M G K K L P L H L T L-

w ~. 1~40'~41
-29-
BS BSS
M SC HS D M H SCAHM
N TR AT D N P TRUAN
L NF EU E L H NF9EL
1 11 31 1 1 1 11631
/ / / /
TGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGA
961 --_______,_________,_________,_________,_________,_________, 1020
ACGGGGTCCGGAACGGAGTCATACGACCGAGACCTTTGGAGTGGGACCGGGAACTTCGCT
P Q A L P Q Y A G S G N L T L A L E A K-
S BS
F SC H D A
A TR P D L
N NF H E U
1 11 1 1 1
AAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGA
1021 --_______,_________,_________,_________,_________,_________, 1080
TTTGTCCTTTCAACGTAGTCCTTCACTTGGACCACCACTACTCTCGGTGAGTCGAGGTCT
T G K L H Q E V N L V V M R A T Q L Q K-
PS S
M ADNNPA DF AM DE A
N VRLLW DA LN DS L
L AAAAM9 EN UL EP U
1 224416 11 11 11 1
///// / / /
AAAAT1'TGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCTTGAAAC
1081 - _ _ _,_________,_________,_________,_________,_________,
- --- - 1140
TTTTAAACTGGACACTCCACACCCCTGGGTGGAGGGGATTCGACTACGACTCGAACTTTG
N L T C E V W G P T S P K L M L S L K L-
M T H M DM
N A P N DS
L Q .A L ET
1 1 2 1 12
TGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGCCGGTGTGGGTGCTGAACCCTG
1141 -________,_________,_________,_________,_________,_________, 1200
ACCTCTTGTTCCTCCGTTTCCAGAGCTTCGCCCTCTTCGGCCACACCCACGACTTCGGAC
E N K E A K V S K R E K P V W V L N P E

- 1340741
H PS H
F D M I A ADPA I
0 D A N V VRUU N
K E E F A AAM9 F
1 1 3 1 1 2216 1
AGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTGGAATCCAACA
1201 _________,_________,_________,_________,_________,_________, 1260
TCCGCCCCTACACCGTCACAGACGACTCACTGAGCCCTGTCCAGGACGACCTTAGGTTGT
A G M W Q C L L S D S G Q V L L E S N I-
S SA BHF BS H
ANA HNCP SGNMAANXA RSD I A
VLU PCRA PIUNMULHV SCD N L
AA9 AIFL lADLH3A0A AAE D U
236 2111 21211A421 111 3 1
TCAAGGTTCTGCCCACATGGTCCACCCCGGTGCACGCGGATCCCGAGGCTGAGTACTAAG
1261 _________,_________,_________,_________,_________,_________, 1320
AGTTCCAAGACGGGTGTACCAGGTGGGGCCACGTGCGCCTAGGGCTCCCACTCATGATTC
K V L P T W S T P V H A D P E
BS g
H H SC HS S M M D S
P A TR AT T N N D P
H E NF EU Y L L E M
1 3 11 31 1 1 1 1 1
CT1TCTGGGGCAGGCCAGGCCTGACCTTGGCTTTCGCGCAGGGAGGGGGCTAAGGTGAGG
1321 _________,_________,_________,_________,_________,_________, 1380
GAAAGACCCCGTCCGGTCCGGACTGGAACCGAAACCCCGTCCCTCCCCCGATTCCACTCC
B A BH g p
BASHBHHNN P SG N BS F H
AHPHBAPAL A PI L AP L G
NAMAEEHRA L lA A N1 M A
12111:?114 1 21 3 22 1 1
I
CAGGTGGCGC(:AGCAGGTGCACACCCAATGCCCATGAGCCCAGACACTGGACGCTGAACC
1381 _________,..________,_________,_________,_________,_________, 1440
GTCCACCGCGGTCGTCCACGTGTGGGTTACGGGTACTCGGGTCTGTGACCTGCGAC1TGG
F BS S B SS B S FN
N M SC DNHA H SMAAHNABSAC NS
U N TR RLAU H TNUUALPAPLR UP
D L NF AAE9 A NL99EAAN1UF pg
2 1 11 2436 1 11663412211 22
TCGCGGACAGT'TAAGAACCCAGGGGCCTCTGCGCCTGGGCCCAGCTCTGTCCCACACCGC
1441 _________,_.________,_________,_________,_________,_________, 1500
AGCGCCTGTCAATTCTTGGGTCCCCGGAGACGCCGACCCGGGTCGAGACAGGGTGTGGCG

1~40'~41
-31-
F BSS BS
MS BNN NM S BMDMHNABSAA SCB
AA ALL UN T BBRNALPAPUU TRA
EC NAA 4L Y VOALEAAN199 NFN
32 134 H1 1 12213412266 111
GGTCACATGCCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATCCGTCTTCCCCCTGG
1501 __ ____~..________,_________;_________,___-_____,_________, 1560
CCAGTCTACCGTGGTGGAGAGAACGTCGGAGGTGGTTCCCGGGTAGCCAGAAGGGGGACC
A S T K C P S V F P L A-
BH B NFS BS F BS
N M MSG MSB SNAH SC N SC
L N NPI NPB PUUA TR U TR
A L L1A LiV B49E NF 4 NF
4 1 121 121 2H63 11 H 11
CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT
1561 -________,..________,_________~____ _,_________,_________, 1620
GTGGGAGGAGGTTCTCGTGGAGACCCCCGTCTCGCCGGGACCCGACGGACCAGTTCCTGA
P S S K S T S G G T A A L G C L V K D Y-
NF A BH
H M T H D BANHBHN SN P SG
P A T P D AHAHBAL PU A PI
A E H H E NARAEEA B4 L lA
2 3 1 1 1 1211124 2H 1 21
ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACA
1621 -________,_________,_________,_________,_________,_________, 1680
TGAAGGGGCTTCGCCACTGCCACAGCACCTTGAGTCCGCGGGACTGGTCGCCGCACGTGT
F P E P V T V S W N S G A L T S G V H T-
S H F B
HNC DM I M D N M SM B
PCR DS N N D U N TA B
AIF ET F L E 4 L EE V
211 12 1 1 1 H 1 23 1
CCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGC
1681 -________,_,________,_________,_________,_________,_________, 1740
GGAAGGGCCGACAGGATGTCAGGAGTCCTGAGATGAGGGAGTCGTCGCACCACTGGCACG
F P A 'V L Q S S G L Y S L S S V V T V P -
B F B B H
SH N ASM B NSB M I
PP U LTN A LPB A N
iH 4 UXt_ N A1V E F
21 H ll:l 1 421 2 1
CCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACA
1741 _________,__._______f_________,_________,_________,_________, 1800
GGAGGTCGTCGAACCCGTCGGTCTGGATGTAGACGTTGCACTTAGTGTTCGGGTCGTTGT
S S S L. G T Q T Y I C N V N H K P S N T -

-32- 13 4 0 7 41
S M HM HM
T N AN PN
Y L EL HL
1 1 31 11
CCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAA
1801_________,____ __,_________,_________,_________,_________,1860
GGTTCCACCTGTTCTTTCAACCACTCTCCGGTCGTGTCCCTCCCTCCCACAGACGACCTT
K V D K K V
E BS SS F BS F
DE CHH F SC HHNCF N BSC N
DS OHA 0 TR PGCRA U BTR U
EP 4AE K NF AAIFN 4 VNF 4
11 712 1 11 21111 H 111 H
/ / // //
GCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCA
1861-________,_________,_________,_________,_________,_________,1920
CGTCCGAGTCGCGAGGACGGACCTGCGTAGGGCCGATACGTCGGGGTCAGGTCCCGTCGT
S S
DBHMHNA HMNCN M MNDM
RBABPLU PNCRL N NLDB
AVEOHA9 ALIFA L LAEO
2132146 21114 1 1312
// // //
AGGCAGGCCCCGTCTGCCTCTTCACCCGGAGCCTCTGCCCGCCCCACTCATGCTCAGGGA
1921-________,_________,_________+_________,_________,_________,1980
TCCGTCCGGGGCAGACGGAGAAGTGGGCCTCGGAGACGGGCGGGGTGAGTACGAGTCCCT
BS P B gS
SC F M B N S SC
TR L A A L P TR
NF M E N A 1 NF
11 1 1 1 4 2 11
A
A
GAGGGTCTTCTGGCTTTTTCCC
GGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCC
1981-________,_________,_________,_________~_________,_________,2040
CTCCCAGAAGACCGAAAAAGGGTCCGAGACCCGTCCGTGTCCGATCCACGGGGATTGGGT
S B B B S
DHA S DBS S M HNC A
RAU P DAP P N PCR V
AE9 M EN1 M L AIF A
236 1 122 1 1 211 2
C
C
G
GG
CCTGCAGACAAAGGGCCAGGTGCTGGGCT
AGACCTCCCAAGAGCCATATCC
GGAG
2041_________,_________,_________,_________,_________,_________,2100
CCGGCACGTGTGTTTCCCCCTCCACGACCCGAGTCTGGACGGTTCTCGGTATAGGCCCTC

-- 134041
PS -33-
DNPA D H D A M
RLW D A D L N
AAM9 E E E U L
2416 1 3 1 1 1
/ //
GACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGA
2101 ________._,____.____,_________,_________,__ _,_________, 2160
CTGGGACGGGGACTGGATTCGGGTGGGGTTfCCGGTTTGAGAGGTGAGGCAGTCGAGCCT
H B
I M MM P BS
N N AB S AP
F L EO T N1
1 1 32 1 22
CACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCT
2161 -________.,_________,_________,_________,_________,_________, 2220
GTCGAAGAGAGGACGGTCTAAGGTCATTGAGGGTTAGAACAGAGACGTCTCGGCTTTAGA
E P K S -
N BBS BS
M NS SSC SC HS M
A LP PTR TR AT N
E AH iNF NF EU L
3 31 211 11 31 1
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCTAAGCCACCCCAGGCCTCGCCCTCC
2221 -________*_________,_________,_________,_________,_________, 2280
ACACTCTTI'TGAGTGTGTACGGGTCGCACGGGTCCATTCGGTCGGGTCCGGAGCGGGAGG
C D K T H T C P P C P
B BS S S S
A M B N SM F SC F DHNA HNC
L N A L PA 0 TR A RALU PCR
U L N A lE K NF N AEA9 AIF
1 1 1 4 21 1 11 1 2346 211
AGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCACGGACAGGCCCCAGCCGGG
2281 -________,~_________,_________,_________i_________,_________, 2340
TCGAGTTCCGCCCTGTCCACGGGATCTCATCGGACGTAGGTCCCTGTCCGGGGTCGGCCC
BS S
A M M M D M SC M ANA M
F A B N D N TR B VLU B
L E 0 L E L NF 0 AA9 0
3 2 2 1 1 1 il 2 246 2
TGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGT
2341 --_______*_________,_________,_________,_________,_________, 2400
ACGACTGTGCAGGTGGAGGTAGAGAAGGAGTCGTGGACTTGAGGACCCCCCTGGCAGTCA
A P E L L G G P S V -

-- I34Q7~~
-34- S SS
M S AN M HMANNAC DM M
N T UL N PNVCLUR DS A
L Y 3A L ALAIA9F ET E
1 1 A3 1 2121461 12 3
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC
2401 _________,_________,_________,_________,_________,_________, 2460
GAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAGTACTAGAGGGCCTGGGGACTCCAGTG
F L F P P K P K D T L M I S R T P E V T -
N
NS M M DM M RM M
LP A N DS B SA N
AH E L ET 0 AE L
31 2 1 12 2 12 1
/ /
ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA
2461 -________,_________,_________,_________,__-______,_______-_, 2520
TACGCACCACCACCTGCACTCGGTGCTTCTGGGACTCCAGTTCAAGTTGACCATGCACCT
C V V 'V D V S H E D P E V K F N W Y V D -
F FN
M N NSS R M R
N U UPA S _~A S
L 4 DBC A E A
1 H 222 1 2 1
//
CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA
2521 -________,_________,_________,_________,_________,_________, 2580
GCCGCACCTCCACGTATTACGGTTCTGTTTCGGCGCCCTCCTCGTCATGTTGTCGTGCAT
G V E 'V H N A K T K P R E E Q Y N S T Y -
S BS
HNC HH M SC R
PCR GP N TR S
AIF AH L NF A
211 11 1 11 1
/ /
CCGGGTCGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
2581 _________,._________,_________,_________,_________,_________, 2640
GGCCCACCAGTCGCAGGAGTGGCAGGACGTGGTCCTGACCGACTTACCGTTCCTCATGTT
R V V S V L T V L H Q D W L N G K E Y K -
M T
N A
L Q
1 1
GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
2641 -________,._________,_________,_________,_________,_._______, 2700
CACGTTCCAGAGGTTGT1'TCGGCAGGGTCGGGGGTAGCTCTTTTGGTAGAGGTTTCGGTT
C K V S N K A L P A P I E K T I S K A K -

~- I34a741
-35-
PS S S
ADNNPMA A H M N HHN BSAH
VRLI_UNU U A N L APA GFUA
AAAAML9 9 E L A EAE LI9E
2244116 6 3 1 3 321 1163
//// /
AGGTGGGACCCGTGCGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTG
2701 -_______..,_________,_________,__ _,_________,_________, 2760
TCCACCCTGGGCACCCCACGCTCCCGGTGTACCTGTCTCCGGCCGAGCCGGGTGGGAGAC
N F
D M M S R M N A g
D N A P S N U V B
E L E B A L 4 A V
1 1 3 2 1 1 H 1 1
CCCTGAGAGTGACCGCTGTACCAACCTCTGTCCTACAGCGCAGCCCCGAGAACCACAGGT
2761 -________,_________,_________,__ _,_________,_________, 2820
GGGACTCTCACTGGCGACATGGTTGGAGACAGGATGTCCCGTCGGGGCTCTTGGTGTCCA
G Q P R E P Q V _
SS BS BS
R F AHNNCCS A F SC SC
S 0 VPCCRRM L 0 TR TR
A K AAIIFFA U K NF NF
1 1 1211111 1 1 11 11
GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCACGTCAGCCTGACCTGCCT
2821 -________.,_________,_________,_________,_________,_________, 2880
CATGTGGGAI:GGGGCTAGGGCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGACGGA
Y T L P P S R D E L T K N Q V S L T C L -
F
S N H
P U P
4 A
1 H 2
GGTCAAAGGCTTCTATCCCAGCGACATCGCCCTGGAGTGGGAGAGCAATGGGCAGCCGGA
2881 _________,_________,_________,_________,_________,_________, 2940
CCAGTTTCCGAAGATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCT
V K G F Y P S 0 I A V E W E S N G Q P E -
H
MI M N H
NN B L P
V LF 0 A H
1 1 1 2 4 1
GAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCCTCCTTCTTCCTCTACAG
2941 _________,_________,_________,_________,_________,_________, 3000
CTTGTTGATGTTCTGGTGCGGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATGTC
N N Y K T T P P V L D S D G S F F L Y S -

.-.,
-36- i 3 4 0 7 41
B F S
M A S NM MBX NF M
N L P UB ABM LA N
L U M 40 EVN AN L
1 1 1 H2 211 31 1
CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT
3001 -________,_.________,_________,_________,_________t_________, 3060
GTTCGAGTGGC:ACCTGTTCTCGTCCACCGTCGTCCCCTTGCAGAAGAGTACGAGGCACTA
K L T V D K S R W Q Q G N V F S C S V M -
S
N N M M HNC
S L B N PCR
I A 0 L AIF
1 3 2 1 211
GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATG
3061 -________,_________,_________,_________,_________,_________~ 3120
CGTACTCCGAGACGTGTTGCTGATGTGCGTCTTCTCGGAGAGGCACAGAGGCCCATTTAC
H E A L. H N H Y T Q K S L S L S P G K
CXHHN
FMAPA
RAEAE
13321
AGTGCGACGGCCG
3121 ----_----~--- 3133
TCACGCTGCCGGC

13~~'~~1
-37-
F N Table 2 S B
N S B M H DHA S
U P B N G RAU T
4 B V L A AE9 X
H 2 1 1 1 236 1
GCCTGTTTGAGAAGCAGCGGGCAAGAAAGACGCAAGCCCAGAGGCCCTGCCATTTCTGTG
1 _________,_________,_________,_________,_________,_________, g0
CGGACAAACTCTTCGTCGCCCGTTCTTTCTGCGTTCGGGTCTCCGGGACGGTAAAGACAC
B PS S S
DBS ADNPA D DHNA M HM HNC
DAP VRLIX! D RALU N AN PCR
EN1 AAAM9 E AEA9 L EL AIF
122 22416 1 2346 1 31 211
/ / /~ / / /
GGCTCAGGTC:CCTACTCGCTCAGGCCCCTCCCTCCCTCGGCAAGGCCACAATGAACCGGG
61 ---______,._________,_________,_________,_________,_________, 120
CCGAGTCCAGGGATGACCGAGTCCGGGGACGGAGGGAGCCGTTCCGGTGTTACTTGGCCC
M N R G -
H F F
I B N HH N M D
N B U HA U N D
F V 4 AE 4 L E
1 1 H 12 H 1 1
GAGTCCCTT1'TAGGCACTTGCTTCTCGTGCTGCAACTGGCCCTCCTCCCAGCAGCCACTC
121 -________,_________,_________,_________,_________,_________, 180
CTCAGGGAAAATCCGTGAACGAAGACCACGACGTTGACCGCGAGGAGGGTCGTCGGTGAG
V P F R H L L L V L Q L A L L P A A T Q-
B E E R A
B C C S L
V 0 0 A U
1 K K 1 1
AGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGCTT
181 -________,;_________,_________,_________,_________,_________, 24C
TCCCTT'TCTIrTCACCACGACCCGTTTTTTCCCCTATGTCACCTTGACTGGACATGTCGAA
G K K V V L G K K G D T V E L T C T A S -
H
M M I
8 B N
0 0 F
2 2 1
CCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAA
241 _________,._________,_________,_________,_________,_________, 300
GGGTCTTCTTCTCGTATGTTAAGGTGACCTTT~'TGAGGTTGGTCTATTTCTAAGACCCTT
Q K K S I Q F H W K N S N Q I K I L G N -

- 1340'41
B S S F H
NBS F AA A A N H I
LAP 0 VU L U U H N
AN1 K A9 U 3 D A F
4 22 1 26 1 A 2 1 1
ATCAGGGC'TCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAA
301 -_______._,_________,_________,_________,_________,_________, 360
TAGTCCCGAGGAACAATTGATT1'CCAGGTAGCTTCCACTTACTAGCGCGACTGAGTTCTT
Q G .~ F L T K G P S K L N D R A D S R R -
S S H H
MANAS BA I A I D
BVLUT CU N F N D
OAA9Y L3 F L F E
22461 lA 1 2 1 1
/ /
GAAGCCTTfGGGACCAACGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACT
361 --______..,_________,_________,_________,_________,_________, 420
CTTCGGAAACCCTGGTTCCTTTGAACGGCGACTAGTAGTTCTTACAATTCTATCTTCTGA
S L Y~ D Q G N F P L I I K N L K I E D S -
S
M M AMAM M
B N VNUN A
0 L AL9L E
2 1 2161 1
//
CAGATACTTACATCTCTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCG
421 -________.,_________,_________,_________,_________,_________, 480
GTCTATGAATGTAGACACTTCACCTCCTGGTCTTCCTCCTCCACGTTAACGATCACAAGC
D T y' I C E V E D Q K E E V Q L L V F G -
B
S S
P T
M Y
1 1
GATTGACTCCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGG
481 -________,_________,_________,_________,_________,_________, 540
CTAACTGACGGTTGAGACTGTGGGTGGACGAAGTCCCCGTCTCGGA('.TGGGACTGGAACC
L T A N S D T H L L Q G Q S L T L T L E -
B BS H
BS SC D M I S
AP TR D N N T
N1 NF E L F Y
22 11 1 1 1 1
/ /
AGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATAC
541 -____-___,_________,_________,__ _,__ _,__ -f 600
TCTCGGGGGGACCATCATCCGGGAGTCACGTTACATCCTCAGGTTCCCCATTTTTCTATG
S P P G S S P S V Q C R S P R G K N I Q -

1340741
-39-
N BBH S B BS
M MD ASP A BSSGSC S B N SC
B ND LPV L APTIAR T A L TR
0 LE UBU U N1NACF X N A NF
2 11 122 1 221111 1 1 4 11
AGGGGGCGAAGACCCTCTCC~TGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACAT
601 -________,_________,_________,_________,_________,__-__-___, 660
TCCCCCCCTTCTGGCAGAGGCACAGAGTCGACCTCGAGGTCCTATCACCGTGGACCTGTA
G G K T L S V S Q L E L Q D S G T W T C-
N
NS M NM A
LP B HA L
AH 0 EE U
31 2 11 1
GCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTT
661 __-______,___-____-,________-,_________,_-_-___-_,__--__-__, 720
CGTGACAGAACGTCTTGGTCTTCTTCCACCTCAAGTTTTATCTGTAGCACCACGATCGAA
T V L Q N Q K K V E F K I D I V V L A F-
HS M M
AT N N
EU L L
31. 1 1
/
TCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCC
721 _________,._________,_________,____-____,_________,___-_____, 780
AGGTCTTCCGGAGGTCGTATCAGATATTCTTTCTCCCCCTTGTCCACCTCAAGAGGAAGG
Q K A S S I V Y K K E G E Q V E F S F P -
A A M
L L N
U U L
1 1 1
CACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGGCGGAGA
781 -________,_________,_________,_________,_- _,_________, 840
GTGAGCGGAA,ATGTCAACTTTCGACTGCCCGTCACCGCTCGACACCACCGTCCGCCTCT
L A F T V E K L T G S G E L W W Q A E R_
P S
H M FM A M
P N LN U g
H L ML 3 0
1 1 11 A 2
GGGCTTCCTCCTCCAACTCTTGGATCACCTTTGACCTGAAGAACAAGGAAGTGTCTGTAA
841 _________,..__-_____,_-_______,_-_______,____-_-__,________-, 900
CCCGAAGGAGGAGGTTCAGAACCTAGTGGAAACTGGACTTCTTGTTCCTTCACAGACATT
A S S S K S W I T F D L K N K E V S V K -

134U741
-40-
B BS PS
SM SCADNPAD A A H
TA TRVRLUUD L L P
EE NFAAAM9E U U H
23 11224161 1 1 1
/ / / //
AACGGGTTACCCAGCACCCTAAGCTCCAGATGGGCAAGAAGCTCCCGCTCCACCTCACCC
901 ~________,_________,_________,_________,_________,_________, 960
TTGCCCAATGGGTCCTGGGATTCGAGGTCTACCCGTTCTTCGAGGGCGAGGTGGAGTGGG
R V T Q D P K L Q M G K K L P L H L T L-
BS BSS
M SC HS D M H SCAHM
N TR AT' D N P TRUAN
L NF EU E L H NF9EL
1 11 31 1 1 1 11631
TGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGA
961 _________,_________,_________,_________,_________,_________,
1020
ACGGGGTCCGGAACGGAGTCATACGACCGAGACCTTTGGAGTGGGACCGGGAACTTCGCT
P Q A L P Q Y A G S G N L T L A L E A K-
S BS
F SC H D A
A TR P D L
N NF H E U
1 11 1 1 1
AAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGA
1021 -________,_.________,_________,_________,_________,_________, 1080
TTTGTCCTTTC:AACGTAGTCCTTCACTTGGACCACCACTACTCTCGGTGAGTCGAGGTCT
T G K L H Q E V N L V V M R A T Q L Q K
PS S
N ADNNPA DF AM DE A
VRLLUU DA LN DS L
L AAAAM9 EN UL EP U
1 224416 11 11 11 1
AAAATTfGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCTTGAAAC
1081 -___ ___,_________,_________,_________,_________,_________, 1140
TTTTAAACTGGACACTCCACACCCCTGGGTGGAGGGGATTCCACTACGACTCGAACTTTG
N L T C E V W G P T S P K L M L S L K L-
M T H M DM
N A P N DS
L Q A L ET
1 1 2 1 12
TGGAGAACAAGGAGGCAAAGGTCTCGAAGCGCGAGAAGCCGGTGTGGGTGCTGAACCCTG
1141 -________+_________,_________,_________,_________,_________, 1200
ACCTCTTGTTCCTCCGTTTCCAGAGCTTCGCCCTCTTCGGCCACACCCACGACTTGGGAC
E N K E A K V S K R E K P V W V L N P E -

-41-
H PS H
F D M I A ADPA I
0 D A N V VRUU N
K E E F A AAM9 F
1 1 3 1 1 2216 1
AGCCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGCTCCTGCTGGAATCCAAU
1201 ________..,_________,_________,_________,_________,_________t 1260
TCCGCCCCTACACCGTCACAGACGACTCACTGAGCCCTGTCCAGGACGACCTTAGGTTGT
A G M W Q C L L S D S G Q V L L E S N I-
S SA BHF BS H
ANA HNCP SGNMAANXA RSD I A
VLU PCRA PIUNMULHV SCD N L
AA9 AIFL lADLH3A0A AAE D U
236 2111 21211A421 111 3 1
TCAAGGTTCTGCCCACATGGTCCACCCCGGTGCACGCGGATCCCGAGGGTGAGTACTAAG
1261 -________.~_________,_________,_________t_________,_________~ 1320
AGTTCCAAGACGGGTGTACCAGCTGCGGCCACGTGCGCCTAGGGCTCCCACTCATGATTC
K V L. P T W S T P V H A D P E
E BS SS F BS F
H CHH F SC HHNCF ~N BSC N
P OHA 0 TR PGCRA U BTR U
H 4AE K NF AAIFN 4 VNF 4
1 712 1 11 21111 H 111 H
CTTCAGCGC'fCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCAAGGC
1321 _________.,_________,_________,_________,_________,_________, 1380
GAAGTCGCGAGGACGGACCTGCGTAGGGCCGATACGTCGGGGTCAGGTCCCGTCGTTCCG
S S
DBH1'HNA N!lNCN M MNDM
RB.4BPLU PNCRL N NLDB
AVEOHA9 A~IFA L LAEO
2132146 21114 1 1312
AGGCCCCGTCTGCCTCTTCACCCGGAGCCTCTCCCCGCCCCACTCATGCTCAGGGAGAGG
1381 -________,_________,_________,_________,_________,_________, 1440
TCCGGGGCAGACGGAGAAGTGGGCCTCGGAGACGGGCGGGGTGAGTACGAGTCCCTCTCC
BS P B BS S
SC F M B N S SCDHA
TR L A A L P TRRAU
NF M E N A 1 NFAE9
11 1 1 1 4 2 11236
GTCTTCTGGCTTTTTCCCAGGCTCTGGGCACGCACAGCCTAGGTGCCCCTAACCCAGGCC
1441 -________~_________,_________,_________+_________,_________, 1500
CAGAACACCGAAAAAGGGTCCGAGACCCGTCCGTGTCCGATCCACCGGGATTGGGTCCGG

1340'~4~.
S PS
S DBS S M HNC ADNPA
P DAP P N PCR VRLUU
M EN1 M L AIF AAAM9
1 122 1 1 211 22416
CTGCACACAAAGGGGCAGGTGCTGGCCTCAGACCTCCCAAGAGCCATATCCGGCAGGACC
1501 -________.,_________,_________,_________,___--__--~____-__-_t 1560
GACGTGTCT'TTCCCCGTCCACGACCCGAGTCTGCACGGTTCTCGGTATAGGCCCTCCTCG
D H D A M
D A D L N
E E E U L
1 3 1 1 1
CTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACC
1561 -________,_________,_________,___ ___,_________,_________, 1620
GACGGGGACTGGATTCGGGTGGGGTTTCCGGTTTGAGAGGTGAGGGAGTCGAGCCTGTGG
H B
I M MM P BS M
N N AB S Ap A
F L EO T N1 E
1 1 32 1 22 3
/ /
T1CTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTG
1621 _________~._________~_________,_________,_________,_________, 1680
AAGAGAGGA(iGGTCTAAGGTCATTGAGCGTTAGAAGAGAGACGTCTCGGGTTTAGAACAC
E P K S C D -
N BBS BS
NS SSC SC HS M A
LF PTR TR AT N L
AH 1N~ NF EU L U
3i 211 11 31 1 1
/ /
ACAAAACTCACACATGCCCA~rGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCT
1681 -________,_________,_________,_________,________
_,_________, 1740
TGTTTTGAGTGTGTACGGGTGGCACGGGTCCATTCGGTCGGGTCCGGAGCGGGAGGTCCA
K T H T C P P C P
BS S S S
M B N SM F SC F DHNA HNC
N A L PA~ 0 TR A RALU PCR
L N A lE K NF N AEA9 AIF
1 1 4 21 1 11 1 2346 211
CAAGGCGGGACAGGTGCCCTAGAGTACCCTGCATCCAGGGACAGGCCCCAGCCGGGTCCT
1741 _________,..________,_________,_________,_________,_________, 1800
GTTCCGCCCTGTCCACGGGATCTCATCGCACGTAGGTCCCTGTCCGCGGTCGGCCCACGA

134Q?~~
-43-
BS S
A M M M D M SC M ANA M
F A B N D N TR B VLU B
L E 0 L E L NF 0 AA9 0
3 2 2 1 1 1 11 2 246 2
/ /
GACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC
1801 _________+_________+_________,_________,_________,_________, 1860
CTGTGCAGGTGGAGGTAGAGAAGGAGTCGTGGACTTGAGGACCCCCCTGGCAGTCAGAAG
A P E L L G G P S V F -
S SS N
M S AN M HMANNAC DM M NS
N T UL N PNVCLUR DS A LP
L Y 3A L ALAIA9F ET E AH
1 1 A3 1 2121461 12 3 31
/ // / /
CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
1861 _________,..________,_________,_________+_________+_________, 1920
GAGAAGGGGGGTTTTGGGTTCCTGTCGGAGTACTAGAGGGCCTGGGGACTCCAGTGTACG
L F P P K P K D T L M I S R T P E V T C -
M M DM M RM M
A N DS B SA N
E: L ET 0 AE L
2 1 12 2 12 1
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGC
1921 --_______,_.________,_________,_________,_________~_________, 1980
CACCACCACC1'GCACTCGGTGCTTCTGGGACTCCAGTTCAAGTTGACCATGCACCTGCCG
V V V D V S H E D P E V K F N W Y V D G -
F FN S
h~ ~' NSS R M R HNC
U UPA S A S PCR
L ~ DBC A E A AIF
1 H 222 1 2 1 211
// /
GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGG
1981 -________,_________,_________i_________,_________~_________, 2040
CACCTCCACGTATTACGGTTCTGTTTCGGCGCCCTCCTCGTCATGTTGTCGTGCATGGCC
V E V H N A K T K P R E E Q Y N S T Y R -

-44- 13 4 0 7 41
BS
HH M SC R
GP N TR S
AH L NF A
11 1 11 1
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
2041 _________,..________,_________+_________,_________,_________, 2100
CACCAGTCGCAGGAGTGGCAGGACGTGGTCCTGACCCACTTACCGTTCCTCATGTTCACG
V V S V L T V L H Q D W L N G K E Y K C -
M T
N A
L Q
1 1
AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGT
2101 -________,_________;_________~_________,_________,_________, 2160
TTCCAGAGGTfGTTTCGGGAGGGTCGGGGGTAGCTCTTTTGGTAGAGGTTTCGGTTTCCA
K V S N K A L P A P I E K T I S K A K
PS S S
ADNNPMA A H M N HHN BSAH p
VRLLUNU U A N L APA GFUA D
AAAAML9 9 E L A EAE LI9E E
2244116 6 3 1 3 321 1163 1
GGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGCCCGGCTCGGCCCACCCTCTGCCCT
2161 -________,_________,_________;_________,_________,_________, 2220
CCCTGGGCACCCCACGCTCCCGGTGTACCTGTCTCCGGCCGAGCCGGGTGGGAGACGGGA
N F
M M S R M N A B R F
N A P S N U V B S 0
L E B A L 4 A V A K
1 3 2 1 1 H 1 1 1 1
GAGAGTGACCGCTGTACCAACCTCTGTCCTACAGGGCAGCCCCGAGAACCACAGGTGTAC
2221 -________,_________,_________,_________,_________,_________, 2280
CTCTCACTGGCGACATGGTTGGAGACAGGATGTCCCGTCGGGGCTCTTGGTGTCCACATG
G Q P R E P Q V Y -

1340'41
- -45-
SS BS BS B
AHNNCCS A F SC SC S
VPCCRR~: L 0 TR TR P
AAIIFFA U K NF NF M
1211111 1 1 11 11 1
ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
2281 -________,._________,_________,_________,_________,______-__, 2340
TGGGACGGGGGTAGGGCCCTACTCGACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAG
T L P P S R D E L T K N Q V S L T C L V -
F
N H B
U P B
4 A V
H 2 1
AAAGGCTTCTATCCCAGCGACATCGCCGTGCAGTGGGAGAGCAATCGGCAGCCGGACAAC
2341 -________,_________,_________,_________,_________,_________, 2400
TTTCCGAAGATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTG
K G F Y P S D I A V E W E S N G Q P E N -
H
MI M N H MA
NN B L P NL
LF 0 A H LU
1 1 2 4 1 1 1
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAG
2401 -________,_________,_________,_________,_________,_________, 2460
TTGATGTTCTGGTGCGGAGGGCACGACCTGAGGCTGCCGAGCAAGAAGGAGATGTCCTTC
N Y K T T P P V L D S D G S F F L Y S K -
B F S
S NM MBX NF M N
~P UB ABM LA N S
M 40 EVN AN L I
1 H2 211 31 1 1
CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTCTTCTCATGCTCCGTGATGCAT
2461 -________,._________,_________,_________,_________,_________, 2520
GAGTGGCACC'tGTTCTCGTCCACCGTCGTCCCCTTGCAGAAGAGTACGACGCACTACGTA
L T V D K S R W Q Q G N V F S C S V M H -

'"' -46-
1340'41
s
N M M HNC
L B N PCR
A 0 L AIF
2 1 211
GAGGCTCTGC~ACAACCACTACACGCAGp,AGAGCCTCTCCCTGTCTCCGGGTAAATGAGTG
2521 -________,._________,_________,_________,________ ______ ?580
_+__ _,
CTCCGAGACG~TGTTGGTGATGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATTTACTCAC
E A L H N H Y T Q K S L S L S P G K
CXH
FMA
RAE
133
CGACGGCCG
2581 -________ ;0589
GCTGCCGGC
TTTCCGAAGATAGGGTCGCT

'" -47-
1340'~41
Table 3
FN S B
N S B M H DHA S
U p B N G RAU T
4 B V L A AE9
H 2 1 1 1 236 1
GCCTGTTTGAGAAGCAGCGGGCAAGAAAGACGCAAGCCCAGAGGCCCTGCCATTTCTGTG
1 _________t..________t_________t_________t_________t_________t 60
CGGACAAACT(:TTCGTCGCCCGTTCTTTCTGCCTTCGGGTCTCCGGGACGGTAAAGACAC
B PS S S
DBS ADNPA D DHNA M HM HNC
DAP VRLUU D RALU N AN pCR
EN1 AAAMSI E AEA9 L EL AIF
122 2241E~ 1 2346 1 31 211
GGCTCAGGTCC:CTACTGGCTCAGGCCCCTGCCTCCCTCGGCAAGGCCACAATGAACCGGG
61 -________t_________t_________t_________t_________t_________t 120
CCGAGTCCAGGGATGACCGAGTCCGGGGACGGAGGGACCCGTTCCGGTGTTACTTGGCCC
M N R G -
H F F
I B N HH N M D
N B U HA U N D
F V 4 AE 4 L E
1 1 H 12 H 1 1
GAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCCCTCCTCCCAGCAGCCACTC
121 _________t_________t_________t_________t_________t_________t 180
CTCAGGGAAAATCCGTGAACGAAGACCACGACGTTGACCGCGAGGAGGGTCGTCGGTGAG
V P F R H L L L V L Q L A L L P A A T Q -
B E E R A
B C C S L
V 0 0 A U
1 K K 1 1
AGGGAAAGAAAI;TGGTGCTGGGCAAAAAAGCGGATACAGTGGAACTGACCTGTACAGCTT
181 -________t_.._______t_________t_________t__ _t__ -t 240
TCCCTTTCTTT(:ACCACGACCCGTTTTTTCCCCTATGTCACCTTGACTGGACATGTCGAA
G K K 11 V L G K K G D T V E L T C T A S -

1340'41
-48-
H
N N
N
0 0 F
2 2 1
CCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAA
241 -________,__._______,_________,_____-___,_________,_________, app
GGGTCTTCTTC1'CGTATGTTAAGGTGACCTTTTTGAGGTTGGTCTATTTCTAAGACCC1?
Q K K S I Q F H W K N S N Q I K I L G N -
B S S F H
NBS F AA A A N H I
LAP 0 VU L U U H N
AN1 K A9 U 3 D A F
422 1 26 1 A 2 1 1
ATCAGGGCTCC'TTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAA
301 -________,__._______,_________,_________,_________,_________, 360
TAGTCCCGAGGA,AGAATTCATTTCCAGGTACGT'fCGACTTACTAGCGCGACTGAGTTCTT
Q G S F' L T K G P S K L N D R A D S R R -
S S H H
MAN,AS BA I A I D
BVLUT CU N F N D
OAA9Y L3 F L F E
22461 lA 1 2 1 1
/ /
GAAGCCTT'TGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACT
361 -________,_________,_________,_________,_________,_________, 420
CTTCGGAAACCCTGGTTCCTTTGAAGGGGGACTAGTAGTTCTTAGAATTCTATCTTCTGA
S L W D Q G N F P L I I K N L K I E D S -
S
M M AMAM M
B N VNUN A
0 L AL9L E
2 1 2161 1
//
CAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGACGTGCAATfGCTAGTGTTCG
421 -________,_________,_________,_________,_________,_________, 4g0
GTCTATGAATGTAGACACTTCACCTCCTGGTCTTCCTCCTCCACGTTAACGATCACAAGC
D T Y I C E V E D Q K E E V Q L L V F G-

I340'~41
'~ -49-
B
S S
P T
M Y
1 1
GATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGG
481 _________,_________,_________,_________,_________,_________, 540
CTAACTGACGGTTGAGACTGTGGGTGGACGAAGTCCCCGTCTCGGACTGGGACTGGAACC
L T A N S D T H L L Q G Q 5 L T L T L E -
B BS H
BS SC D ~' I S
AP TR D N N T
N1 NF E L F Y
22 11 1 1 1 1
AGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATAC
541 -________,_________,_-_______,_________,_________,_________, 600
TCTCGGGGGGACCATCATCGGGGAGTCACGTTACATCCTCAGGTTCCCCATTI'TTGTATG
S P P G S S P S V Q C R S P R G K N I Q-
N BBH S B BS
M MD ASP A BSSGSC S B N SC
B ND LPV L APTIAR T A L TR
0 LE UBU U N1NACF X N A NF
2 11 122 1 221111 1 1 4 11
// / /// /
AGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACAT
601 _________,_________,_________,_________,_________,_________, 660
TCCCCCCCTTCTGGGAGAGGCACAGAGTCGACCTCGAGGTCCTATCACCGTGGACCTGTA
G G K T L S V S Q L E L Q D S G T W T C -
N
NS M NM A
LP B HA L
AH 0 EE U
31 2 11 1
/
GCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTT
661 -________,_________,_________,_________,_________,_________, 720
CGTGACAGAACGTCTTGGTCTTCTTCCACCTCAAGT1TTATCTGTAGCACCACGATCGAA
T V L Q N Q K K V E F K I D I V V L A F-

.~.
-50-
HS M M
AT N N
EU L L 41
1340'
31 1 1
TCCAGAAGG(:CTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCC
721-________,._________,_________,_________,_________,_________,780
AGGTCTTCCGGAGGTCGTATCAGATATTCTTTCTCCCCCTTGTCCACCTCAAGAGGAAGG
Q K A S S I V Y K K E G E Q V E F S F P -
A A M
L L N
U U L
1 1 1
CACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGGCGGAGA
781-________,._________,_________,_________,_________t________
-t 840
GTGAGCGGAAATGTCAACTTTTCGACTGCCCGTCACCGCTCGACACCACCGTCCGCCTCT
L A F T V E K L T G S G L H H' Q A E R -
P S
H M FN A M
P N LN U g
H L ML 3 p
1 1 11 A 2
GGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAAGTGTCTGTAA
841-________,_________,_________,_________+_________,_________,900
CCCGAAGGAGGAGGTTCAGAACCTAGTGGAAACTGGACTTCTTGTTCCTTCACAGACATT
A S S S K S W I T F D L K N K E V S V K -
B BS PS
SM SCADNPAD A A H
TA TRVRLUUD L L P
EE NFAAAM9E U U H
23 11224161 1 1 1
/ / / //
AACGGGT1ACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCGCTCCACCTCACCC
901-________i_________,_________,_________,_________,_________,960
TTGCCCAATGGGTCCTGGGATTCGAGCTCTACCCGTTCTTCGAGGGCGAGGTGGAGTGGG
R V T Q D P K L Q M G K K L P L H L T L -
BS BSS
M SC HS D M H SCAHM
N TR AT D N P TRUAN
L NF EU E L H NF9EL
1 11 31 1 1 1 11631
A
C
T
C
TGCCCC
GG
CTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCC
GG
CCTTGAAGCGA
961-________,_________,_________,_________,_________,_________+1020
ACGGGGTCCGGAACGGAGTCATACGACCGAGACCTTTGGAGTGGCACCGGGAACTTCGCT
P Q A L P Q Y A G S G N L T L A L E A K -

...
-51- 1340741.
S BS
F SC H D A
A TR P D L
N NF H E U
1 11 1 1 1
AAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGA
1021 -________,,_________,_________,_________,_________,_________, 1080
TTTGTCCTlTCAACGTAGTCCTTCACTTGGACCACCACTACTCTCGGTGAGTCGAGGTCT
T G K. L H Q E V N L V V M R A T Q L Q K
PS S
M ADNNPA DF AM DE A
N VRLLUU DA LN DS L
L AAAA~~ 9 EN UL EP U
1 224415 11 11 11 1
///// / / /
AAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCTTGAAAC
1081 -________,_________,_________,__ _,_________,__ -. 1140
TTTTAAACTGGACACTCCACACCCCTGGGTGGAGGGGATTCGACTACGACTCGAACTTTG
N L T C E V W G P T S P K L M L S L K L -
M T H M DM
N A P N DS
L Q A L ET
1 1 2 1 12
TGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGCCGGTGTGGGTGCTGAACCCTG
1141 -________,_________,_________,_________,__ _,_______-_; 1200
ACCTCTTGTTCCTCCGTTTCCAGAGCTTCGCCCTCTTCGGCCACACCCACGACTTGGGAC
E N K E A K V S K R E K P V W V L N P E -
H PS H
F D M I A ADPA I
0 D A N V VRUU N
K E E F A AAMg F
1 1 3 1 1 2218 1
AGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTGGAATCCAACA
1201 -________,,_________,_________,_________,_________f_________t 1260
TCCGCCCCTACACCGTCACACACGACTCACTCAGCCCTGTCCAGGACGACCTTAGGTTGT
A G M W Q C L L S D S G Q V L L E S N I-

.-~,
52 1340741
S SA BHF BS H
ANA HNCP SGNMAANXA RSD I A
VLU PCRA PIUNMULHV SCD N L
AA9 AIFL lADLH3A0A AAE D U
236 2111 21211A421 111 3 1
// // / / / / /
TCAAGGTTCTGCCCACATGGTCCACCCCGGTGCACGCGGATCCCGAGGGTGAGTACTAAG
1261_________,_________,_________,_________,_________,_________,1320
AGTTCCAAGACGGGTGTACCAGGTGGGGCCACGTGCGCCTAGGGCTCCCACTCATGATTC
K V L P T W S T P V H A 0 P E
E BS SS F BS F
H CHH F SC HHNCF N BSC N
P OHA 0 TR PGCRA U BTR U
H 4AE K NF AAIFN 4 VNF 4
1 712 1 11 21111 H 111 H
A
CTTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCC
GGGCAGCAAGGC
1321-________~_________,_________._________w_________._________,1380
GAAGTCGCGAGGACGGACCTGCGTAGGGCCGATACGTCGGGGTCAGGTCCCGTCGTTCCG
S
DBHMHNA HNNCN A~ ~,~NJ;~
RBABPLU PNCR- N N~DB
AVEOHA9 ALIFA L LAtO
2132146 21114 1 1312
// // //
AGGCCCCGTCTGCCTCTTCACCCGGAGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGG
1381-________,_________,_________,_________~_________,_________,1440
TCCGGGGCAGACGGAGAAGTGGGCCTCGGACACGGGCGGGGTGAGTACGAGTCCCTCTCC
BS P B BS S
SC F M B N S SCDHA
TR L A A L P TRRAU
NF M E N A 1 NFAE9
11 1 1 1 4 2 11236
A
A
C
GTCTTCTGGCTT1TTCCC
GGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCC
CG
C
1441-________.,_________,_________,_________+_________,_________t1500
CAGAAGACCGAAAAAGGGTCCGAGACCCGTCCGTGTCCGATCCACGGGGATTGGGTCCGG
B B B S PS
S DBS S M HNC ADNPA
P DAP P N PCR VRLUU
M EN1 M L AIF AAAM9
1 122 1 1 211 22416
G
CTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCC
GGAGGACC
1501-________.,_________,_________,_________;_________,_________,1560
GACGTGTGTTTCCCCGTCCACGACCCGAGTCTGGACGGTTCTCCGTATAGGCCCTCCTGG

_ 1340741
H D A M
D A D L N
t. E E U L
3 1 1 1
CTGCCCCTGAC(:TAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACC
1561 -________;_..__-____,_________+_________,_________,_________, 1620
GACGGGGACTCGATTCGGGTGGGGTTTCCGGTTTGAGAGGTGAGGGAGTCGAGCCTGTGG
L P L T * A H P K G Q T L H S L S S D T -
C P * P K P T P K A K L S T P S A R T P -
A P D 1. S P P Q R P N S P L P Q L G H L -
H S
I M MM pF F
N N AB DO A
F L EO EK N
1 1 32 11 1
/ /
TTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTCAGGGAGTGCATCCGCCCC
1621 _________,_________,_________,_________~_________,_________, 1680
AAGAGAGGAGGGTCTAAGGTCATTGAGGGTTAGAAGAGAGAGTCCCTCACGTAGGCGGGG
G S A S A P -
F_
N
N 0
L R
1 1
AACCCTTT'TCCCCCTCGTCTCCTGTGAGAATTCC....
1681 -________+_________,_________+____ 1714
TTGGGAAAAGGGGGAGCAGAGGACACTCTTAAGG....
T L F P L V S C E N S ....

-54-
1344'41
Table 4
FN S g
N S B M H DHA S
U P B N G RAU T
4 B V L A AE9 X
H 2 1 1 1 236 1
GCCTGTTTGAGAAGCAGCGGGCAAGAAAGACGCAAGCCCAGAGGCCCTGCCATTTCTGTG
1 _________,..________;_________,_________;_________;_________, 60
CGGACAAACTCTTCGTCGCCCGTTCTTfCTGCGTTCGGGTCTCCGGGACGGTAAAGACAC
B PS S S
DBS ADNPA D DHNA M HM HNC
DAP VRLUU D RALU N AN PCR
EN1 AAAM51 E AEA9 L EL AIF
122 2241E~ 1 2346 1 31 211
GGCTCAGGTCC:CTACTGCCTCAGGCCCCTGCCTCCCTCGGCAAGGCCACAATGAACCGGG
61 --_______f_.________;_________;_________;_________;___._____; 120
CCGAGTCCAGGGATGACCGAGTCCGGGGACGGAGGGAGCCGTTCCGGTGTTACTTGGCCC
M N R G -
H F F
I B N HH N M D
N B U HA U N D
F V 4 AE 4 L E
1 1 H 12 H 1 1
GAGTCCCTTTfAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGCAGCCACTC
121 - __,,_________;_________;_________,_________;_________, 180
CTCAGGGAAAATCCGTGAACGAAGACCACGACGTTGACCGCGAGGAGGGTCGTCGGTGAG
V P F R H L L L V L Q L A L L P A A T Q -
B E E R A
B C C S L
V 0 0 A U
1 K K 1 1
AGGGAAAGAAAGTGGTGCTGGCCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGCTT
181 -________,_________;_________;_________;__ _;_________, 240
TCCCTTTCTTTCACCACGACCCGTTTTTTCCCCTATGTCACCTTGACTGGACATGTCGAA
G K K 'V V L G K K G D T V E L T C T A S -

-55- 1340741
H
M M I
B B N
0 0 r
2 2 1
CCCAGAAGp,AGAGCATACAATTCCACTGGAAAAACTCCAACCA~ATAAAGATTCTGGGAA
241 --_______+_________+_________,_________,_________+_________+ 30G
GGGTCTTCTTCTCGTATGTTAAGGTGACCTTTTTGAGGT'TGGTCTATTTCTAAGACCCTT
Q K K S I Q F H W K N S N Q I K I L G N -
B S S F H
NBS F AA A A N H I
LAP 0 VU L U U H N
AN1 K A9 U 3 D A F
422 1 26 1 A 2 1 1
/ /
ATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAA
301 --_______;_________+_________+_________+_________+_________+ 360
TAGTCCCGAGGAAGAATTGATTTCCAGGTAGGTTCGACTTACTAGCGCGACTGAGTTCTT
Q G S F L T K G P S K L N D R A D S R R -
S S H H
MANAS BA I A I D
BVLUT CU N F N D
OAA9Y L3 F L F E
22461 lA 1 2 1 1
GAAGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACT
361 -________,_________,_________+_________+_________+_________+ 420
CTTCGGAAACCCTGGTTCCTTTGAAGGGGGACTAGTAGTTCTTAGAATTCTATCTTCTGA
S L W D Q G N F P L I I K N L K I E D S-
S
M M AMAM M
B N VNUN A
0 L AL9L E
2 1 2161 1
//
CAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGCTGCAATTGCTAGTGTTCG
421 --_______.,___-_____+______-__,________-+_________~________
-+ 480
GTCTATGAA'fCTAGACACTTCACCTCCTGGTCTTCCTCCTCCACGTTAACGATCACAAGC
D T Y I C E V E D Q K E E V Q L L V F G-

.~
- 1340'~4~.
B
S S
' P T
M Y
1 1
GATTGAC'fGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGG
481 -____._.._,_________,__ _,__ _,_________,_________, 540
CTAACTGACGGTTGAGACTGTGGGTGGACGAACTCCCCGTCTCGGACTGGGACTGGAACC
L T A N S D T H L L Q G Q S L T L T L E -
B BS H
BS SC D N I S
AP TR D N N T
N1 NF E L F Y
22 11 1 1 1 1
/ /
AGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATAC
541 -________,_________,_________,___ _,_________,_________, 600
TCTCGGGGGGACCATCATCGGGGAGTCACGTTACATCCTCAGGTTCCCCATTTTTGTATG
S P P G S S P S V Q C R S P R G K N I Q -
N BBH S B BS
M MD ASP A BSSGSC S B N SC
B ND LPV L APTIAR T A L TR
0 LE UBU U N1NACF X N A NF
2 11 122 1 221111 1 1 4 11
AGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACAT
601 -________,_________,_________,_________,_________+_________, 660
TCCCCCCCTTCTGGGAGAGGCACAGAGTCGACCTCGAGGTCCTATCACCGTGGACCTGTA
G G K T L S V S Q L E L Q D S G T W T C -
N
NS M NM A
LP B HA L
AH 0 EE U
31 2 11 1
GCACTGTC~TTGCAGAACCAGAAGAAGGTGGAGT'TCAAAATAGACATCGTGGTGCTAGCTT
661 _________,_________,_________,_________,_________,_________, 720
CCTGACAGAACGTCTTGGTCTTCTTCCACCTCAAGTTI'TATCTGTAGCACCACGATCGAA
T V IL Q N Q K K V E F K I D I V V L A F -
HS M M
.AT N N
IEU L L
31 1 1
TCCAGAAGGCCTCCAGCATAGTCTATAAGAAACAGGGGCAACAGGTGGAGTTCTCCTTCC
721 -_______._,_________,_________,_________,_________,_________, 780
AGGT(TTCCGGAGGTCGTATCAGATATTCTTTCTCCCCCTTGTCCACCTCAACAGGAACG
Q K A S S I V Y K K E G E Q V E F S F P-

-57-
1340741
A A
L L N
U U L
1 1 1
CACTCGCCT'T'fACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGGCGGAGA
781 -________.,_________y_________y_________,_________,_________,840
GTGAGCGGAAATGTCAACT~TTTCGACTGCCCGTCACCGCTCGACACCACCGTCCGCCTCT
L A F' T V E K L T G S G E L w H' Q A E R -
P S
H M FM A M
P N LN U g
H L ML 3 p
1 1 11 A 2
GGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAAGTGTCTGTAA
841 -________y_________y_________y_________,_________y_________y900
CCCGAAGGAGGAGGTTCAGAACCTAGTGGAAACTGGACTTCTTGTTCCTTCACAGACATT
A S S. S K S W I T F D L K N K E V S V K -
B BS PS
SM SCADNPAD A A H
TA TRVRLWD L L P
EE NFAAAM9E U U H
23 11224161 1 1 1
/ / / //
AACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCGCTCCACCTCACCC
901 -________y_________y_________,_________,_________y_________y960
TTGCCCAATGGGTCCTGGCATTCGAGGTCTACCCGTTCTTCGAGGGCGAGGTGGAGTGGG
R V 1' Q D P K L Q M G K K L P L H L T L -
BS BSS
M SC HS D M H SCAHM
N TR AT D N P TRUAN
L NF EU E L H NF9EL
1 il 31 1 1 1 11631
/ / / /
TGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCCCT?GAAGCGA
961 -________.y_________y_________,_________,_________y_________y1020
ACGGGGTCC:GGAACGGAGTCATACGACCGAGACCTTTGGAGTGCGACCGGGAACTTCGCT
P Q A L P Q Y A G S G N L T L A L E A K -
S BS
F SC H D A
A TR P D L
N NF H E U
1 il 1 1 1
AAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGA
1021 -________;_________y_________,_________,_________y_________,1080
TTTGTCCTTTCAACGTAGTCCT1CACTTGGACCACCACTACTCTCGGTGAGTCGAGGTCT
T G K L H Q E V N L V V M R A T Q L Q K -

134 0 741
-58-
PS S
M ADNNPA DF AM DE A
N VRLLUU DA LN DS L
L AAAAM9 EN UL EP U
1 224416 11 11 11 1
AAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCiTGAAAC
1081-________,_________,_______ _,_________,__ _,_________,1140
TTTTAAACTGGACACTCCACACCCCTGGGTGGAGGGGATTCGACTACGACTCGAACTTTG
N L T' C E V W G P T S P K L M L S L K L -
M T H M DM
N A P N DS
L Q A L ET
1 1 2 1 12
TGGAGAACAAGGAGGCAAAGGTCTCGAAGCGCGAGAAGCCGGTGTGGGTGCTGAACCCTG
1141-________,_________,_________,_____ _,_________,_________,1200
ACCTCTTGTTCCTCCGTT'TCCAGAGCTTCGCCCTCTTCGGCCACACCCACGACTTGGGAC
E N K E A K V S K R E K P V W V L N P E -
H PS H
F D M I A ADPA I
0 D A N V VRUU N
K E E F A AAN9 F
1 1 3 1 1 2216 1
'
AGGCGGGGA
TGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTGGAATCCAACA
1201_________.,_________,_________,_________,_________,_________,1260
TCCGCCCCTACACCGTCACAGACGACTCACTGAGCCCTGTCCAGGACGACCTTAGGTTGT
A G M W Q C L L S D S G Q V L L E S N I _
S SA BHF BS H
ANA HNCP SGNNAANXA RSD I A
VLU PCRA PIUNMULHV SCD N L
AA9 AIFL lADLH3A0A AAE D U
236 2111 21211A421 111 3 1
// // / / / / /
TCAAGGTTC'TGCCCACATGGTCCACCCCGGTGCACGCGGATCCCGAGGGTGAGTACTAAG
1261-________.,_________,_________,_________,_________,_________,1320
AGTTCCAAGACGGGTCTACCAGGTGGGGCCACGTGCGCCTAGGGCTCCCACTCATGATTC
K V L P T W S T P V H A D P E
E BS SS F BS F
H CHH F SC HHNCF N BSC N
P OHA 0 TR PGCRA U BTR U
H 4AE K NF AAIFN 4 VNF 4
1 712 1 11 21111 H 111 H
CTTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCAAGGC
1321-________,_________,_________,_________,_________,_________.
1380
GAAGTCGCGAGGACGGACCTGCGTAGGGCCGATACGTCGGGGTCAGGTCCCGTCGTTCCG

-59-
1340?41
S s
DBHt~IHNA HMNCN M MND~!
RBABPLU PNCRL N NLRB
AVEOHA9 ALIFA L LAEC
2132146 21114 1 1312
// // //
AGGCCCCGT'CTGCCTCTTCACCCGGAGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGG
1381_________,_________,_________,_________,_____-___,_________,1440
TCCGGGGCA,GACGGAGAAGTGGGCCTCGGAGACGGGCGGGGTGAGTACGAGTCCCTCTCC
BS P B BS S
SC F M B N S SCDHA
TR L A A L P TRRAU
NF M E N A 1 NFAE9
11 1 1 1 4 2 11236
A
GTCTTCTGGCTTTfTCCC
GGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCC
1441-________,_________,_________,_________,_________,_________,1500
CAGAAGACCGAAAAAGGGTCCGAGACCCGTCCGTGTCCGATCCACGGGGATTGGGTCCGG
B B B S PS
S DBS S M HNC ADNPA
P DAP P N PCR VRLUU
M EN1 M L AIF AAAM9
1 122 1 1 211 22416
CTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGACGACC
1501-________,_________,_________,___-_____,_________,_________,1560
GACGTGTGTTTCCCCGTCCACGACCCGAGTCTGGACGGTTCTCGGTATAGGCCCTCCTGG
D H D A M
D A D L N
E E E U L
1 3 1 1 1
CTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCACCTCGGACACC
1561--_______.,_________,_________,_________,_________,_________,1620
GACGGGGAC'fGGATTCGGGTGGGGTTTCCGGTTTGAGAGGTGAGGGAGTCGAGCCTGTGG
H F
I M MM BP DE AN
N N AB BS DS LU
F L EO VT EP U4
1 1 32 11 11 1H
/ / /
TTCTCTCCTCCCAGATTCCAGTAACTCCCAATCT?CTCTCTGCAGTGATTGCTGAGCTGC
1621-________,_________,_________,_________,___._____;__.______,1680
AAGAGAGGAGGGTCTAAGGTCATTGAGGGT'TAGAAGAGAGACGTCACTAACGACTCGACG
V I A E L P -

-60-
1340741
F
M H N M N
B G N B U
D A L 0 D
2 1 1 2 2
CTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAAGT
1681 -________.,_________,_________,_________,_________,_________, 1740
GAGGGTTTC:ACTCGCAGAAGCAGGGTGGGGCGCTGCCGAAGAAGCCGTTGGGGGCGTTCA
P K V S V F V P P R D G F F G N P R K S-
BS S H B S F
A SC H HNC I B SMC N
L TR A PCR N B TNR U
U NF E AIF F V NLF 4
1 11 3 211 1 1 111 H
CCAAGCTCATCTGCCAGGCCACGCGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTGC
1741 -________,_________,_________,_________,_________,_________, 1800
GGTTCGAGTAGACGGTCCGGTGCCCAAAGTCAGGGGCCGTCTAAGTCCACAGGACCGACG
K L I C Q A T G F S P R Q I Q V S W L R _
F B S BS H
NH S H H AM AA SCM D H I
UH P P G HA VU TRN D A N
DA M H A AE A9 NFL E E F
21 1 1 1 ?3 26 111 1 3 1
/ / /
GCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTCAGGCCAAAG
1801 _________,_________,_________,_________,_________,_________, 1860
CGCTCCCCTTCGTCCACCCCAGACCGCAGTGGTGCCTGGTCCACGTCCGACTCCGGTTTC
E G K Q V G S G V T T D Q V Q A E A K E-
SS B 8
AAHNABS SM H
UUALPAP TA P
99EAAN1 EE H
6634122 23 1
/ // /
~AGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGAG....
1861 -________,_________,_________,_________,_________, 1910
TCAGACCCGGGTGCTGGATGTTCCACTCGTCGTGTGACTGGTAGTTTCTC....
S G P T T Y K V T S T l T I K E ....

-6I - 13 4 0'~ 41
Table 5
FN S g
N S B M H DHA S
U P B N G RAU T
4 B V L A AE9 X
H 2 1 1 1 236 1
GCCTGTTTGAGAAGCAGCGGGCAAGAAAGACGCAAGCCCAGAGGCCCTGCCATTTCTGTG
1 ____.___.,_________,________ ______
_,_________,__ _,_________, g0
CGGACAAACTCTTCGTCGCCCGTTCTTTCTGCGTTCGGGTCTCCGGGACGGTAAAGACAC
B PS S S
DBS ADNPA D DHNA M HM HNC
DAP VRLUU D RALU N AN PCR
EN1 AAA~19 E AEA9 L EL AIF
122 22416 1 2346 1 31 211
GCCTCAGGT~CCCTACTGGCTCAGGCCCCTGCCTCCCTCGGCAAGGCCACAATGAACCGGG
gl _________.,_________,_________+_________,_________,_________, 120
CCGAGTCCAGGGATGACCGAGTCCGGGGACGGAGGGAGCCGTTCCGGTGTTACTTGGCCC
M N R G -
H F F
I B N HH N M D
N B U HA U N D
F V 4 AE 4 L E
1 1 H 12 H 1 1
GAGTCCCTTI'TAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGCAGCCACTC
121 -________.,_________,,_________,_________,_________,_________, 180
CTCAGGGAAAATCCGTGAACGAAGACCACGACGTTGACCGCGAGGAGGGTCGTCGGTGAG
V P F R H L L L V L Q L A L L P A A T Q-
B E E R A
B C C S L
V 0 0 A U
1 K K 1 1
AGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTACAGCTT
181 -________,._________,_________,_________,_________,_________, 240
TCCCTTTCTTTCACCACGACCCGTTTII'TCCCCTATGTCACCTTGACTGGACATGTCGAA
G K K V V L G K K G D T V E L T C T A S-
H
M M I
B B N
0 0 F
2 2 1
CCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAA
241 -________,_________,_________,_________,_________,_________, 300
GGGTCTTCTTCTCGTATGTTAAGGTGACCTTTTTGAGGTTGGTCTATTTCTAAGACCCTT
Q K K S I Q F H W K N S N Q I K I L G N -

134074.
-62-
B S S F H
NESS F AA A A N H I
LAP 0 VU L U U H N
AN1 K A9 U 3 D A F
42'2 1 26 1 A 2 1 1
ATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAA
301 _________,_________,_________,_________t_________,_________. 360
TAGTCCCGAGGAAGAATTGATTTCCAGGTAGGTTCGACTTACTAGCGCGACTGACTTCTT
Q G S F L T K G P S K L N D R A D S R R_
S S H H
MANAS BA I A I D
BVLUT CU N F N D
OAA9Y L3 F L F E
22461 lA 1 2 1 1
GAAGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAGATAGAAGACT
361 -________,_________,_________,_________,_________,_________, 420
CTTCGGAAACCCTGGTTCCTTTCAAGGGGGACTAGTAGTTCTTAGAATTCTATCTTCTGA
S L W D Q G N F P L I I K N L K I E D S-
S
M M AMAM M
B N VNUN A
0 L AL9L E
2 1 2161 1
CAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCG
421 _________.,_________,_________,_________,_________f_________, 480
GTCTATGAATGTAGACACTTCACCTCCTGGTCTTCCTCCTCCACGTTAACGATCACAAGC
D T Y I C E V E D Q K E E V Q L L V F G-
B
S S
P T
M Y
1 1
GATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGG
481 -________,_________,_________,_________,_________,_________, 540
CTAACTGACGGTTGAGACTGTGGGTGGACGAAGTCCCCGTCTCGGACTGGGACTGGAACC
L T A N S 0 T H L L Q G Q S L T L T L E-

B BS 63 H 13 4 0 '~ 41
BS SC D M I S
AP TR D N N T
N1 NF E L F Y
22 11 1 1 1 1
/ /
AGACCCC(:CCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATAC
541 -_______._,_________,_________,_________,_________,_________, 600
TCTCGGGCGGACCATCATCGGGGAGTCACGTTACATCCTCAGGTTCCCCATTTTTGTATG
S P P G S S P S V Q C R S P R G K N I Q-
N BBH S B BS
M MD ASP A BSSGSC S B N SC
B ND LPV L APTIAR T A L TR
0 LE UBU U N1NACF X N A NF
2 11 122 1 221111 1 1 4 11
AGGGGGGGAAGACCCTCTCCGTGTCTCACCTGGAGCTCCAGGATAGTGGCACCTGGACAT
601 -_______._,_________,_________,_________,_________,_________, 660
TCCCCCCC:TTCTGGGAGAGGCACAGAGTCGACCTCGACGTCCTATCACCGTGGACCTGTA
G G K T L S V S Q L E L Q D S G T W T C-
N
NS M NM A
LP B HA L
AH 0 EE U
31 2 11 1
GCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTACCTT
661 -________~_________,_________,_________,________-;_________, 720
CGTGACAGAACGTC1TGGTCTTCTTCCACCTCAAGTTTTATCTGTAGCACCACGATCCAA
T V L Q N Q K K V E F K I D I V V L A F -
HS M M
AT N N
EU L L
31 1 1
TCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGCGAACACGTGGAGTTCTCCTTCC
721 -_______._,_________,_________,_________,_________,_________, 780
AGGTCTTC:CGGAGGTCGTATCAGATATTCTTTCTCCCCCTTGTCCACCTCAAGAGGAAGG
Q K A S S I V Y K K E G E Q V E F S F P -
A A M
L L N
U U L
1 1 1
CACTCGCC.TTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGGCGGAGA
781 --_______,-________,_________,_________,_________,_________, 840
GTGAGCGGAAATGTCAACTTTTCGACTCCCCGTCACCGCTCGACACCACCGTCCGCCTCT
L A F T V E K L T G S G E L W W Q A E R _

-64-
1340741
P S
H M FM A M
P N LN U B
H L ML 3 p
1 1 11 A 2
GGGCTTCCT(:CTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAAGTGTCTGTAA
841 _________~._________,______-__,_._.___._,_____._._,__.______, 900
CCCGAAGGAGGAGGTTCAGAACCTAGTGGAAACTGGACTTCTTGTTCCTTCACAGACATT
A S S S K S W I T F D L K N K E V S V K-
B BS PS
SM SCADNPAD A A H
TA TRVRLUUD L L p
EE NFAAAM9E U U H
23 11224161 1 1 1
AACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCGCTCCACCTCACCC
901 -________,_________,_________,__ ____,_________,_________, 960
TTGCCCAATCGGTCCTCGGATTCGACGTCTACCCCTTCTTCGAGGGCGAGGTGGAGTGGG
R V T Q D P K L Q M G K K L P L H L T L -
BS BSS
M SC HS D M H SCAHM
N TR AT D N P TRUAN
L NF EU E L H NF9EL
1 11 31 1 1 1 11631
TGCCCCAGGC(:TTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGA
961 _________,..________,_________,__ _,_________,_________, 1020
ACGGGGTCCGGAACGGAGTCATACGACCGAGACCTTTGGAGTGGGACCGGGAACTTCGCT
P Q A L P Q Y A G S G N L T L A L E A K -
S BS
F SC H D A
A TR P D L
N NF H E U
1 11 , 1 1 1
AAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGA
1021 -_-______,_________,_________,________.,______.._,__._____., 1080
TTTGTCCTTTCAACGTAGTCCTTCACTTGGACCACCACTACTCTCGGTGAGTCGAGGTCT
T G K L H Q E V N L V V M R A T Q L Q K_

-65- 1~40'~4i
PS S
M ADNNPA DF AM DE A
N VRLLUU DA LN DS L
L AAAAM9 EN UL EP U
1 224416 11 11 11 1
///// / / /
AAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCTTGAAAC
1081 -________i_________,_________~_________,_________+_________; 1140
TTTTAAACTGGACACTCCACACCCCTGGGTGGAGGGGATTCGACTACGACTCGAACTTTG
N L T C E V W G P T S P K L M L S L K L-
N T H M DM
N A P N DS
L Q A L ET
1 1 2 1 12
TGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGCCGGTGTGGGTGCTGAACCCTG
1141 -________,_________,_________,_________,_________;_________, 1200
ACCTCTTGTTCCTCCGTTTCCAGAGCTTCGCCCTCTTCGGCCACACCCACGACTTGGGAC
E N K E A K V S K R E K P V W V L N P E -
H PS H
F D M I A ADPA I
0 D A N V VRUU N
K E E F A AAM9 F
1 1 3 1 1 2216 1
///
AGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGCACAGGTCCTGCTGGAATCCAACA
1201 -________,,_________,~_________,_____-___,_________f___-_____, 1260
TCCGCCCCTACACCGTCACAGACGACTCACTGAGCCCTGTCCAGGACGACCTTAGGTTGT
A G M W Q C L L S D S G Q V L L E S N I-
S SA BHF BS B
ANA HNCP SGNMAANXA SH
VLU PCRA PIUNMULHV PP
AA9 AIFL lADLH3A0A 1H
236 2111 21211A421 21
// // / / / /
TCAAGGTTCTGCCCACATGGTCCACCCCGGTGCACGCGGATCCCGAGGGTGAGTGTGCCC
1261 -________;..________;_________,_________,_________+_________, 1320
AGTTCCAAGACGGGTGTACCAGGTGGGGCCACGTGCGCCTAGGGCTCCCACTCACACGGG
K V L P T W S T P V H A D P E

-66- 13 4 0'~ 41
BS S S S
MF SC F DHNA HNC A M M
AO TR A RALU PCR F A B
EK NF N AEA9 AIF L E 0
11 11 1 2346 211 3 2 2
/ / / /
TAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCT
1321 -_______._,_________,_________,_________,_________,_________, 1380
ATCTCATCGGACGTAGGTCCCTGTCCGGCGTCGGCCCACGACTGTGCAGGTGGAGGTAGA
BS S
M D M SC M ANA M M S
N D N TR B VLU B N T
L E L NF 0 AA9 0 L Y
1 1 1 11 2 246 2 1 1
CTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
1381 -________,_________,_________,_________,_________,_________, lt4C
GAAGGAGTCGTGGACTTGAGGACCCCCCTGGCAGTCACAAGGAGAAGGGGGGTT'TTGGGT
A P E L L G G P S V F L F P P K P K-
S SS N
AN M HMANNAC DM M NS M
UL N PNVCLUR DS A LP A
3A L ALAIA9F ET E AH E
A3 1 2121461 12 3 31 2
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC
1441 -_______._,_________,_________,_________,_________,_________, 1500
TCCTGTGGGAGTACTAGAGCGCCTGGGCACTCCAGTGTACGCACCACCACCTGCACTCGG
D T I_ M I S R T P E V T C V V V D V S H -
M DM M RM M
N DS B SA N
L ET 0 AE L
1 12 2 12 1
ACGAA6AC(:CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
1501 -_______..,_________,_________,_________,_________,_________, 1560
TGCTTCTGGGACTCCAGTTCAAGTTGACCATGCACCTGCCGCACCTCCACGTATTACGGT
E D F' E V K F N W Y V D G V E V H N A K -
F FN S
M N NSS R M R HNC HH
N ll UPA S A S PCR GP
L 4 DBC A E A AIF AH
1 H 222 1 2 1 211 11
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCG
1561 -________.,_________,_________,_________,_________,_________, 1620
TCTGTTTCGGCGCCCTCCTCGTCATGTTGTCGTGCATGGCCCACCAGTCGCAGGAGTGGC
T K P R E E Q Y N S T Y R V V S V L T V-

-67-
1340'41
BS
M SC: R
N TR S
L NF' A
1 11 1
/.
TCCTGCACCA,GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC
1621 _________,_________,_________,_________,_________,_________, 1680
AGGACGTGGTCCTGACCGACTTACCGTTCCTCATGTTCACGTTCCAGAGGT'TGTTTCGGG
L H Q D W L N G K E Y K C K V S N K A L -
PS S
T ADNNPMA A
N A VRLLUNU U
L Q AAAAML9 9
1 1 2244116 6
////
TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAG
1681 -________,_________,_________,_________,_________,_________, 1740
AGGGTCGGGGGTAGCTCTTTTGGTAGAGGTTTCGGTTTCCACCCTCGGCACCCCACGCTC
P A P I E K T I S K A K
S N
H M N HHN BSAH D M M S R
A N L APA GFUA D N A P S
E L A EAE LI9E E L E B A
3 1 3 321 1163 1 1 3 2 1
GGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCA
1741 -________,_________,_________,_________,_________,_________, 1800
CCGGTGTACCTGTCTCCGGCCGAGCCGGGTGGGAGACGGGACTCTCACTGGCGACATGGT
F SS
M N A B R F AHNNCC
N U V B S 0 VPCCRR
L 4 A V A K AAIIFF
1 H 1 1 1 1 121111
////
ACCTCTGTCCTACAGGGCAGCCCCCAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG
1801 -________,_________,_________,_________,_________,_________, 1860
TGGAGACAGGATGTCCCGTCGGGGCTCTTGGTGTCCACATGTGGGACGGGGGTAGGGCCC
G Q P R E P Q V Y T L P P S R D-

_ -68-
BS gS g 13 4 0'~ 41
S A F SC SC S
M L 0 TR TR P
A U K NF NF M
1 1 1 11 11 1
ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCG
1861 -________,..________,_________,_________,_________,_________, 1920
TACTCGACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTfCCGAAGATAGGGTCGC
E L T K N Q V S L T C L V K G F Y P S D-
F
N H B
U P B
4 A V
H 2 1
ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTC
1921 -________i..________,_________,_________+_________+_________, 1980
TGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATGTTCTGGTGCGGAG
I A V E H' E S N G Q P E N N Y K T T P P -
H g
A" I M N H M A S
NN B L P NL P
L F 0 A H L U M
1 1 2 4 1 1 1 1
CCGTGCTGGAC:TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCA
1981 -________,_.________,_________t_________,_________,_________. 2040
GGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATGTCGTTCGAGTGGCACCTGTTCTCGT
V L D S D G S F F L Y S K L T V D K S R-
F S
NM MBX NF M N N
UB ABM LA N S L
40 EVN AN L I A
H2 211 31 1 1 3
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
2041 -________,_.________,_________~_________,_________,_________, 2100
CCACCGTCGTC:CCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGAGACGTGTTGGTGA
W Q Q G N V F S C S V M H E A L H N H Y -
S
M M HNC CXH
B N PCR FMA
0 L AIF RAE
2 1 211 133
/ /
ACACCCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCG
2101 -________,_________,_________t_________,_________, 2150
TGTGCGTCTTCTCGGAGAGGGACAGAGGCCCATTTACTCACGCTGCCGGC
T Q K S L S L S P G K

-69- 13 4 0'~ 41
Example 2: Preparation of the Fusion Proteins from Supernatants of
COS Cells
COS cells grown in DME medium supplemented with 10% Calf Serum
and gentamicin sulfate at 15 ~tg/ml were split into DME medium
containing 10% NuSerum (Collaborative Research) and gentamicin to give
50% confluence the day before transfection. The next day, CsCI
purified plasmid DNA was added to a final concentration of 0.1 to 2.0
ug/ml followed by DEAE Dextran to 400 ug/ml and chloroquine to 100 ~M.
After 4 hours at 37°C, the medium was aspirated and a 10% solution
of
dimethyl sulfnxide in phosphate buffered saline was added for 2
minutes, aspirated, and replaced with DME/10% Calf Serum. 8 to 24
hours later, the cells were trypsinized and split 1:2.
For radio'labeling, the medium was aspirated 40 to 48 hours after
transfection, the cells washed once with phosphate buffered saline,
and DME medium lacking cysteine or methionine was added. 30 minutes
later, 35S-labeled cysteine and methionine were added to final
concentrations of 30-60 ~ci and 100-200 ~ci respectively, and the
cells allowed to incorporate label for 8 to 24 more hours. The
supernatants were recovered and examined by electrophoresis on 7.5%
polyacrylamide gels following denaturation and reduction, or on 5%
polyacrylamide following denaturation without reduction. The CD4B~y1
protein gave the same molecular mass with or without reduction, while
the CD4E~1 and CD4H~y1 fusion proteins showed molecular masses without
reduction of twice the mass observed with reduction, indicating that
they formed dimer structures. The CD4 IgM fusion proteins formed
large multimers beyond the resolution of the gel system without
reduction, and monomers of the expected molecular mass with reduction.
Unlabeled proteins were prepared by allowing the cells to grow
for 5 to 10 days post transfection in DME medium containing 5% NuSerum
and gentamicin as above. The supernatants were harvested,
centrifuged, and purified by batch adsorption to either protein A

1340'41
-~o-
trisacryl, protein A agarose, goat anti-human IgG antibody agarose,
rabbit anti-human IgM antibody agarose, or monoclonal anti-CD4
antibody agarose. Antibody agarose conjugates were prepared by
coupling purified antibodies to cyanogen bromide activated agarose
according to the manufacturer's recommendations, and using an antibody
concentration of 1 mg/ml. Following batch adsorption by shaking
overnight on a rotary table, the beads were harvested by pouring into
a sintered glass funnel and washed a few times on the funnel with
phosphate buffered saline containing 1% N* P40 detergent. The
beads were removed from the funnel and poured into a small disposable
plastic column (Quik-Sep Qsit*column, Isolab), washed with at least 20
column volumes of phosphate buffered saline containing 1% Nonidet P40,
with 5 volumes of 0.15 M NaCI, 1 mM EDTA (pH 8.0), and eluted by the
addition of either 0.1 M acetic acid, 0.1 M acetic acid containing 0.1
M NaCI, or 0.25 M glycine-HC1 buffer, pH 2.5.
example 3' Blockage of Svncvtium Formation by the Fusion Proteins
Purified or partially purified fusion proteins were added to HPB-
ALL cells infected 12 hours previously with a vaccinia virus
recombinant encoding HIV envelope protein. After incubation for 6-8
more hours, the cells were washed with phosphate buffered saline,
fixed with formaldehyde, and photographed. All of the full-length CD4
immunoglobulin fusion proteins showed inhibition of syncytium
formation at a concentration of 20 ~g/ml with the exception of the
4H~1 protein, which was tested only at 5 ~g/ml and showed partial
inhibition of syncytium formation under the same conditions.
~xamole 4: Chromium Release Cvtolvsis Assav
The purified fusion proteins were examined for ability to fix
complement in a chromium release assay using vaccinia virus infected
cells as a model system. Namalwa (B cell) or HPB-ALL (T cell) lines
~,;~~;,x~, *Trademe~rk

_7,_ i34074~.
were infected with vaccinia virus encoding HIV envelope protein, and
18 hours later were radiolabeled by incubation in 1 mci/ml sodium
5lchromate in phosphate buffered saline for 1 hour at 37'. The
labeled cells were centrifuged to remove the unincorporated chromate,
and incubated in microtiter wells with serial dilutions of the CD4
immunoglobulin fusion proteins and rabbit complement at a final
concentration of 40%. After 1 hour at 37', the cells were mixed well,
centrifuged, and the supernatants counted in a gamma-ray counter. No
specific release could be convincingly documented.
Example 5: Binding of the CD4E~1 Protein to Fc Receptors
Purified CD4E~1 fusion protein was tested for its ability to
displace radiolabeled human IgGl from human Fc receptors expressed on
COS cells in culture. The IgGl was radiolabeled with sodium 125iodide
using 1 mci of iodide, 100 ~g of IgGl, and two idobeads (Pierce). The
labeled protein was separated from unincorporated counts by passage
over a sic*G25 column equilibrated with phosphate buffered saline
containing 0.5 mM EOTA and 5% nonfat milk. Serial dilutions of the
CD4E~1 fusion protein or unlabeled IgGI were prepared and mixed with a
constant amount of radiolabeled IgGl tracer. After incubation with
COS cells bearing the FcRI and RcRII receptors at 4'C for at least 45
mi nutes i n a vol ume of 20 ~1, 200 ul of a 3 : 2 mi xture of d i butyl to
dioctyl phthalates were added, and the cells separated from the
unbound label by centrifugation in a microcentrifuge for 15 to 30
seconds. The tubes were cut with scissors, and the cell pellets
counted in a gamma-ray counter. The affinity of the CD4E~1 protein
for receptors was measured in parallel with the affinity of the
authentic IgGl protein, and was found to be the same, within
experimental error.
Example 6: Stable Expression of the Fusion Construct~CD4Ey1 in Babv
Hamster Kidnev Cells
*Trademark

.,~ 1~ 40741
_72_
Twenty-four hours before transfection, 0.5 x 106 baby hamster
kidney cells (BHK; ATCC CCL10) were seeded in a 25 cm2 culture flask
in Dulbecco's modified Eagle's medium (DMEM) containing 10% of fetal
calf serum (FCS). The cells were cotransfected with a mixture of the
plasmids pCD4E~r1 (20 fig), pSV2dhfr (5 ug; Lee et al., Nature 294:228-
232 (1981)) and pRMH140 (5 ug, Hudziak et al., Cell 31:137-146 (1982))
according to a modified calcium phosphate transfection technique as
described in Zettlmeissl et al. (Behrina Inst. Res. Comm. _82:26-34
(1988)). 72 h post-transfection, cells were split 1:3 to 1:4 (60 mm
culture dishes) and resistant colonies were selected in DMEM medium
containing 10i~ FCS, 400 ~cg/ml 6418 (Geneticin, Gibco) and 1 ~M
methotrexate (selection medium). The medium was changed twice a week.
The resistant colonies (40-100/transfection) appeared 10-15 day post-
transfection and were further propagated either as a mixture of clones
(i.e., BHK-MK1) or as individually isolated clones. For the
determination of the relative expression levels, clone mixtures or
individual clones were grown to confluency in T25 culture flasks,
washed twice with protein-free DMEM medium, and incubated for 24 h
with 5 ml protein-free DMEM medium. These media were collected and
subjected to a human IgG specific ELISA in order to determine the
relative expression levels of the CD4-IgGl fusion protein CD4E~1. For
further analysis an individual clone (BHK-UC3) was chosen due to its
high relative expression levels.
Example 7: Detection of the CD4E~1 Protein in Culture Supernatants
For 35S methionine labeling of cells, the clone BHK-UC3 and
untransfected BHK cells (control) were grown to confluency in T25
culture flasks and subsequently incubated for two hours in HamFl2
medium without methionine. Labeling was achieved by incubating 24 h
in 2.5 ml of the same medium containing 100 ~Ci 35S methionine (1070
Ci/mmole, Amersham). For the preparation of cell lysates, the labeled

-73- 134a?41
cells were harvested in 1 ml of phosphate buffered saline, pH 7.2
(PBS) and lysed by repetitive freezing and thawing. Cleared lysates
(after centrifugation x0000 rpm, 20 min) and culture supernatants were
incubated with Protein A-Sepharose (Pharmacia) and the bound material
was analyzed on a 10% SDS-Protein A-Sc~h~r~oee* (Pharmacia) and the
bound material was analyzed on a 10% SDS-gel according to Laemmli
(Nature X7:680-685 (1970)), which was subsequently autoradiographed.
A specific band of about 80 KDa can be detected only in the
supernatant of clone BHK-UC3, which is absent in the lysate of clone
BHK-UC3 and in the respective controls.
Example 8: Purification of the Protein CD4E~1 from Culture
Supernatants
In order to demonstrate that the fusion protein coded by the
plasmid pCD4E~1 can be obtained in high quantities, the clone BHK-UC3
was grown in 1750 cm2 roller bottles in selection medium (500 ml).
Confluent monolayers were washed twice with protein-free DMEM medium
(200 ml) and further incubated for 48 h with protein-free DMEM medium
(500 ml). The conditioned culture supernatants (1-2 1) and respective
supernatants from untransfected BHK cells were cleared by
centrifugation (9000 rpm, 30 min) and microfiltered through a 0.45 um
membrane (Nalgene). After addition of 1% (v/v) of 1.9 M Tris-HCl
buffer, pH 8.6, the conditioned medium was absorbed to a Protein A-
Sepharose column equilibrated with 50 mM Tris-HC1 pH 8.6 buffer
containing 150 mM NaCI (4'C). The loaded column was washed with 10
column volumes of equilibration buffer. flution of the CD4-IgGl
fusion protein CD4E~1 was achieved with 0.1 M sodium citrate buffer,
pH 3, followed by immediate neutralization of the column efflux to pH
8 by Tris-base. The peak fractions were pooled, and the pool was
analyzed on a Coomassie blue stained SDS-gel resulting in a band of
the expected size (80 KDa), and which reacted with a polyclonal anti-
human IgG heavy chain antibody and a mouse monoclonal anti-CD4
~~
Trademark

134074:
- -74-
antibody (BMA040, Behringwerke) in Western Blots. The yields of
purified fusion proteins obtained by the given procedure is 5-18 mg/24
h/1 culture supernatant. The respective value for a BHK clone mixture
(about 80 resistant clones; BHK-MK1) as described above was 2-3 mg/24
h/1.
~xamnle 9: Physical and Biological Characterization of the CD4E~r1
Fusion Protein
As proven by SOS-electrophoresis on 10-15% gradient gels (Phast-
S~Ste~n*, Pharmacia) under non-reductive conditions, the CD4E~1 fusion
protein migrates at the position of a homodimer (about 160 KDa) like a
non-reduced mouse monoclonal antibody. This result is supported by
analytical equilibrium ultracentrifugation, where the fusion protein
behaves as a homogeneous dimeric molecule of about 150 KDa. The
absorbance coefficient of the protein was determined as A2g0 = 18
cm2/mg using the quantitative protein determination according to
Bradford (Anal. io hem. 72:248-254 (1976)).
The CD4Ey1-fusion protein shows specific complex formation with a
solubilized gal-gp120 fusion protein (pMB1790; Broker et al., Behring
Inst. Res. Commun. x:338-348 (1988)) expressed in . coli. In this
protein (110 KDa), a major part of the HIV gp120 protein (Val4g-
Trp646) is fused to ~-galactosidase (amino acids 1-375). In a control
experiment a 67-KDa pgal-HIV 3'orf fusion protein (Sgall-375; 3'orf
Prol4-Asp123) showed noncomplex formation. In these experiments, the
CD4Ey1-protein was incubated with the respective fusion protein in
molar rations of about 5:1. The complex was isolated by binding to
Protein A-Sepharose and the Protein A-Sepharose bound proteins--
together with relevant controls--were analyzed on 10-15% gradient SDS-
gels (Phast-System, Pharmacia).
The CD4E~1 fusion protein binds to the surface of HIV (HIV1/HTLV-
IIIB) infected cultured T4-lymphocytes as determined by direct
immunofluorescence with fluorescein-isothiocyanate (FITC) labeled
*Trademark

-75-
13 4 p'~ 41
CD4E~1 protein. It blocks syncytia formation in cultured T4-
lymphocytes upon HIY infection (0.25 TCID/cell) at a concentration of
~g/ml. furthermore, HIV-infected cultured T4-lymphocytes (subclone
of cell line H9) are selectively killed upon incubation with CD4E~1 in
the presence or absence of complement: To a highly (>50/) HIU
infected culture of T4-lymphocytes (106 cells/ml) 50, 10 or 1 ~g/ml
CD4E~1 fusian protein was added in the presence or absence of guinea
pig complement. Cells were observed for specific killing by the
fusion protein, which is defined by the percentage of killed cells
after 3 days in relation to viable cells in the culture at the
beginning of the experiment corrected by the values for unspecific
killing observed in control cultures, lacking the CD4E~1 fusion
protein (Table 6. Experiment I). Surprisingly, addition of CD4E71
protein to the infected T4 cells in the absence of complement resulted
in similar ;specific killing rates as in the presence of complement
(Table 6. Experiment II). This result demonstrates a complement
independent cytolytic effect of CD4E~1 on HIU infected T-lymphocytes
in culture.
Tabla 6
No. Experiment Assav Svstem Specific Killing (/e)
I non-infected T4-cells 0.7
+ 50 ~g/ml CD4Ey1 + Compl.
infected T4-cells 35.1
+ 50 ~g/ml CD4ET1 + Compl.
infected T4-cells 25.1
+ 10 ~cg/ml CD4Ey1 + Compl.
infected T4-cells 25
+ 1 ug/ml CD4E~1 + Compl.
II infected T4-cells 49.9
+ 10 ug/ml CD4E~1 + Compl.
infected T4-cells 69.4
+ 10 ~g/ml CD4E71 + Compl.

,r
1340'41
Having now fully described this invention, it will be appreciated
by those ski 11 ed i n the art that the same can be performed wi th any
wide range of equivalent parameters of composition, conditions, and
methods of preparing such fusion proteins without departing from the
spirit or scope of the invention or any embodiment thereof.