Claims for Patent: 5,547,853

Summary for Patent: 5,547,853

Title:	CD2-binding domain of lymphocyte function associated antigen 3
Abstract:	Polypeptides and proteins comprising the CD2-binding domain of LFA-3 are disclosed. DNA sequences that code on expression for those polypeptides and proteins, methods of producing and using those polypeptides and proteins, and therapeutic and diagnostic compositions are also disclosed. Deletion mutants unable to bind CD2 and methods for their use are also disclosed. In addition, fusion proteins which comprise the CD2-binding domain of LFA-3 and a portion of a protein other than LFA-3, DNA sequences encoding those fusion proteins, methods for producing those fusion proteins, and uses of those fusion proteins are disclosed.
Inventor(s):	Wallner; Barbara P. (Cambridge, MA), Miller; Glenn T. (Haverhill, MA), Rosa; Margaret D. (Winchester, MA)
Assignee:	Biogen, Inc. (Cambridge, MA)
Application Number:	07/940,861
Patent Claims:	1. A polypeptide having the amino acid sequence: X.sub.1 -X.sub.2 -(SEQ ID NO:1) Asn Arg Val Tyr Leu Asp Thr Val Ser Gly-Y, wherein: X.sub.1 if present, is hydrogen or methionyl; X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy-terminal 1 to 77 amino acids of the sequence (SEQ ID NO:5): Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln Ile Tyr Gly Val val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino terminal 1 to 32 amino acids of the sequence (SEQ ID NO:33): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met Lys Phe Phe Leu Tyr Val; said polypeptide being capable of binding to CD2. 2. The polypeptide according to claim 1 wherein, X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy terminal 1 to 50 amino acids of the sequence (SEQ ID NO:2): Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino-terminal 1 to 10 amino acids of the sequence (SEQ ID NO:3): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser; said polypeptide being capable of binding to CD2. 3. A polypeptide selected from the group consisting of polypeptides having the amino acid sequences: amino acids 29-120 of SEQ ID NO:10, amino acids 29-108 of SEQ ID NO:10, amino acids 48-108 of SEQ ID NO:10, and SEQ ID NO:7. 4. A polypeptide having the amino acid sequence (SEQ ID NO:7): Lys Asn Arg Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr. 5. A multimeric protein capable of binding to CD2 comprising two or more polypeptides according to any one of claims 1-4. 6. A method for labeling CD2.sup.+ cells or proteins containing the LFA-3 binding domain of CD2, said method comprising the step of incubating the CD2.sup.+ cells or proteins containing the LFA-3 binding domain of CD2 with a polypeptide according to any one of claims 1-4. 7. A pharmaceutical composition comprising a polypeptide according to any one of claims 1-4 and a pharmaceutically acceptable carrier. 8. A diagnostic kit for detecting the presence of CD2.sup.+ cells or LFA-3-binding proteins comprising a polypeptide according to any one of claims 1-4 together with one or more reagents necessary for detecting the binding of CD2.sup.+ cells or LFA-3-binding proteins to the polypeptide. 9. An isolated DNA encoding a polypeptide selected from the group consisting of: (a) a polypeptide having the amino acid sequence: X.sub.1 -X.sub.2 -(SEQ ID NO:1) Asn Arg Val Tyr Leu Asp Thr Val Ser Gly-Y, wherein: X.sub.1 if present, is hydrogen or methionyl; X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy-terminal 1 to 77 amino acids of the sequence (SEQ ID NO:5): Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino terminal 1 to 32 amino acids of the sequence (SEQ ID NO:33): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met Lys Phe Phe Leu Tyr Val; said polypeptide being capable of binding to CD2; (b) a polypeptide having the amino acid sequence: X.sub.1 -X.sub.2 -(SEQ ID NO:1) Asn Arg Val Tyr Leu Asp Thr Val Ser Gly-Y, wherein: X.sub.1 if present, is hydrogen or methionyl; X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy terminal 1 to 50 amino acids of the sequence (SEQ ID NO:2): Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino-terminal 1 to 10 amino acids of the sequence (SEQ ID NO:3): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser; said polypeptide being capable of binding to CD2; (c) a polypeptide having the amino acid sequence of amino acids 29-120 of SEQ ID No:10; (d) a polypeptide having the amino acid sequence of amino acids 29-108 of SEQ ID No:10; (e) a polypeptide having the amino acid sequence of amino acids 48-108 of SEQ ID No:10; and (f) a polypeptide having the amino acid sequence of (SEQ ID No:7): Lys Asn Arg Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr. 10. A recombinant DNA molecule comprising a DNA according to claim 9 and an expression control sequence, wherein the expression control sequence is operatively linked to the DNA. 11. A method for producing a CD2 binding protein comprising the steps of culturing a host cell transformed with the recombinant DNA molecule of claim 10 and recovering the protein encoded thereby. 12. A host cell transformed with the recombinant DNA molecule according to claim 10. 13. An isolated DNA encoding the CD2-binding domain of LFA-3 consisting of: (5') (SEQ ID NO:6) AATAGGGTTT ATTTAGACAC TGTGTCAGGT (3'). 14. A recombinant DNA molecule comprising a DNA according to claim 13 and an expression control sequence, wherein the expression control sequence is operatively linked to the DNA. 15. An isolated DNA consisting of a DNA encoding the CD2-binding domain of LFA-3 selected from the group consisting of the DNA inserts of pPYM57, pPMDRM54-6, pPMDRM55-9, pPMDRM56-C, pPMDRM58-15, pPYM63-4, pPYM65-8, pPMDRM100-4, pPMDRM101-1, pPMDRM102-8, and pPMDRM3-10. 16. A recombinant DNA molecule comprising a DNA according to claim 15 and an expression control sequence, wherein the expression control sequence is operatively linked to the DNA. 17. A method for producing a CD2 binding protein comprising the steps of culturing a host cell transformed with the recombinant DNA molecule of claim 14 or 16 and recovering the protein encoded thereby. 18. A host cell transformed with the recombinant DNA molecule according to claim 14 or 16. 19. A fusion protein, having an amino terminal region consisting of the amino acid sequence of a polypeptide selected from the group consisting of; (a) a polypeptide having the amino acid sequence: X.sub.1 -X.sub.2 -(SEQ. ID NO:1) Asn Arg Val Tyr Leu Asp Thr Val Ser Gly-Y, wherein: X.sub.1 if present, is hydrogen or methionyl; X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy-terminal 1 to 77 amino acids of the sequence (SEQ ID NO:5): Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino terminal 1 to 32 amino acids of the sequence (SEQ ID NO:33): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met Lys Phe Phe Leu Tyr Val; said polypeptide being capable of binding to CD2; (b) a polypeptide having the amino acid sequence: X.sub.1 -X.sub.2 -(SEQ. ID NO:1) Asn Arg Val Tyr Leu Asp Thr Val Ser Gly-Y, wherein: X.sub.1 if present, is hydrogen or methionyl; X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy terminal 1 to 50 amino acids of the sequence (SEQ ID NO:2): Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino-terminal 1 to 10 amino acids of the sequence (SEQ ID NO:3): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser; said polypeptide being capable of binding to CD2; (c) a polypeptide having the amino acid sequence of amino acids 29-120 of SEQ ID No:10; (d) a polypeptide having the amino acid sequence of amino acids 29-108 of SEQ ID No:10; (e) a polypeptide having the amino acid sequence of amino acids 48-108 of SEQ ID No:10; and (f) a polypeptide having the amino acid sequence of (SEQ ID No:7): Lys Asn Arg Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr; and having a carboxy terminal region comprising a domain of a protein or polypeptide other than LFA-3. 20. The fusion protein according to claim 19 wherein the carboxy terminal region comprises at least a portion of the Fc region of an immunoglobulin. 21. The fusion protein according to claim 20 wherein the portion of the Fc region of an immunoglobulin comprises a hinge region and C.sub.H 2 and C.sub.H 3 constant domains. 22. The fusion protein according to claim 20 wherein the portion of the Fc region comprises a portion of a hinge region that is capable of forming intermolecular disulfide bonds and C.sub.H 2 and C.sub.H 3 constant domains. 23. The fusion protein according to claim 22 which is amino acids 29-347 of SEQ ID NO:43. 24. An isolated DNA coding for a fusion protein according to any one of claims 19-23. 25. A recombinant DNA molecule comprising the DNA according to claim 24 and an expression control sequence, wherein the expression control sequence is operatively linked to the DNA. 26. A method for producing a CD2 binding protein comprising the steps of culturing a host cell transformed with the recombinant DNA molecule of claim 25 and recovering the protein encoded thereby. 27. A host cell transformed with the recombinant DNA molecule according to claim 25. 28. The recombinant DNA molecule, according to claim 25 selected from the group consisting of plasmid pSAB152 and plasmid pMDR(92)Ig-3. 29. A method for producing a CD2 binding protein comprising the steps of culturing a host cell transformed with the recombinant DNA molecule of claim 28 and recovering the protein encoded thereby. 30. A host cell transformed with the recombinant DNA molecule according to claim 28. 31. A multimeric protein capable of binding to CD2 comprising two or more fusion proteins according to any one of claims 19-23. 32. A method for labeling CD2.sup.+ cells or proteins containing the LFA-3 binding domain of CD2, said method comprising the step of incubating the CD2.sup.+ cells or proteins containing the LFA-3 binding domain of CD2 with a fusion protein according to any one of claims 19-23. 33. A pharmaceutical composition comprising a fusion protein according to any one of claims 19-23, and a pharmaceutically acceptable carrier. 34. A diagnostic kit comprising a fusion protein according to any one of claims 19-23 together with one or more reagents necessary for detecting the binding of CD2.sup.+ cells or LFA-3-binding proteins to the fusion protein. 35. A multimeric protein capable of binding to CD2 comprising: (a) one or more polypeptides having the amino acid sequence: X.sub.1 -X.sub.2 -(SEQ ID NO:1) Asn Arg Val Tyr Leu Asp Thr Val Ser Gly-Y, wherein: X.sub.1 if present, is hydrogen or methionyl; X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy-terminal 1 to 77 amino acids of the sequence (SEQ ID NO:5): Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino terminal 1 to 32 amino acids of the sequence (SEQ ID NO:33): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met Lys Phe Phe Leu Tyr Val; said polypeptides being capable of binding to CD2; and (b) one or more fusion proteins having an amino terminal region consisting of the amino acid sequence of a polypeptide having the amino acid sequence: X.sub.1 -X.sub.2 -(SEQ ID NO:1) Asn Arg Val Tyr Leu Asp Thr Val Ser Gly-Y, wherein: X.sub.1 if present, is hydrogen or methionyl; X.sub.2, if present, is a polypeptide having the following amino acid sequence or a portion thereof consisting of the carboxy-terminal 1 to 77 amino acids of the sequence (SEQ ID NO:5): Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys; Y is hydroxyl or a polypeptide of the following amino acid sequence or a portion thereof consisting of the amino terminal 1 to 32 amino acids of the sequence (SEQ ID NO:33): Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met Lys Phe Phe Leu Tyr Val; said polypeptide being capable of binding to CD2; and having a carboxy terminal region comprising a domain of a protein or polypeptide other than LFA-3.

Details for Patent 5,547,853

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Astellas Pharma Us, Inc.	AMEVIVE	alefacept	For Injection	125036	01/30/2003		2013-08-20

International Patent Family for US Patent 5,547,853

Country	Patent Number	Estimated Expiration
World Intellectual Property Organization (WIPO)	9216622
United States of America	5928643
United States of America	5914111
United States of America	5728677
Singapore	47766
Portugal	503648