# US 5,648,464 A · Human Notch and Delta binding domains in toporythmic proteins, and methods based thereon

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- Number: US 5,648,464 A
- Document ID: US5648464A
- Status: Granted
- Kind: A
- Application number: 08/264,534
- Filed: 1994-06-23
- Granted: 1997-07-15
- Earliest claimed: 1991-05-03
- Assignees: Yale University
- Inventors: Spyridon Artavanis-Tsakonas; Richard Grant Fehon; Ilaria Rebay; Christine Marie Blaumueller
- Web page: https://patentgrep.com/patent/US5648464A

## CPC

- C07K 16/18: against material from animals or humans
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- Y10S 530/839: Nerves; brain
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The present invention relates to nucleotide sequences of the human Notch and Delta genes, and amino acid sequences of their encoded proteins, as well as fragments thereof containing an antigenic determinant or which are functionally active. The invention is also directed to fragments (termed herein "adhesive fragments"), and the sequences thereof, of the proteins ("toporythmic proteins") encoded by toporythmic genes which mediate homotypic or heterotypic binding to toporythmic proteins. Toporythmic genes, as used herein, refers to the genes Notch, Delta, and Serrate, as well as other members of the Delta/Serrate family which may be identified, e.g., by the methods described herein. Analogs and derivatives of the adhesive fragments which retain binding activity are also provided. Antibodies to human Notch and to adhesive fragments are additionally provided. In specific embodiments, the adhesive fragment of Notch is that fragment comprising the Notch sequence most homologous to Drosophila Notch EGF-like repeats 11 and 12; the adhesive fragment of Delta mediating heterotypic binding is that fragment comprising the sequence most homologous to Drosophila Delta amino acids 1-230; the adhesive fragment of Delta mediating homotypic binding is that fragment comprising the sequence most homologous to Drosophila Delta amino acids 32-230; and the adhesive fragment of Serrate is that fragment comprising the sequence most homologous to Drosophila Serrate amino acids 85-283.

## Claims

### Claim 1

A substantially purified protein comprising an amino acid sequence encoded by the DNA sequence depicted in FIG. 19A (SEQ ID NO:13), 19B (SEQ ID NO:14) or 19C (SEQ ID NO:15), which is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 2

A substantially purified protein comprising an amino acid sequence encoded by the DNA sequence depicted in FIG. 20A (SEQ ID NO:16), 20B (SEQ ID NO:17) or 20C (SEQ ID NO:18), or 20D (SEQ ID NO:19), which is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 3

A substantially purified protein comprising an amino acid sequence encoded by the DNA sequence depicted in FIG. 21A (SEQ ID NO:20), or 21B (SEQ ID NO:21), which is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 4

A substantially purified protein comprising an amino acid sequence encoded by the DNA sequence depicted in FIG. 22A (SEQ ID NO:22), 22B (SEQ ID NO:23) or 22C (SEQ ID NO:24), or 22D (SEQ ID NO:25), which is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 5

A substantially purified protein comprising an amino acid sequence encoded by the DNA sequence depicted in FIG. 19A (SEQ ID NO:13), 19B (SEQ ID NO:14), 19C (SEQ ID NO:15), 20A (SEQ ID NO:16), 20B (SEQ ID NO:17), 20C (SEQ ID NO:18), 20D (SEQ ID NO:19), 21A (SEQ ID NO:20), 21B (SEQ ID NO:21), 22A (SEQ ID NO:22), 22B (SEQ ID NO:23), 22C (SEQ ID NO:24), or 22D (SEQ ID NO:25), which is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 6

A substantially purified protein comprising the portion of a human Notch protein with the greatest homology over a sequence of at least 77 amino acids, to the epidermal growth factor-like repeats 11 and 12 of the Drosophila Notch sequence as shown in FIG. 8 (SEQ ID NO:1), in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO: 34).

### Claim 7 (depends on 1)

A chimeric protein comprising the protein of claim 1 joined to a heterologous protein sequence.

### Claim 8

A substantially purified protein comprising: a fragment of a human Notch protein consisting of at least 77 amino acids, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 9

A substantially purified protein comprising: a fragment of a human Notch protein consisting essentially of the extracellular domain of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 10

A substantially purified protein comprising: a fragment of a human Notch protein consisting essentially of the intracellular domain of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 11

A substantially purified protein comprising: a fragment of a human Notch protein consisting essentially of the extracellular and transmembrane domains of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 12

A substantially purified protein comprising: a fragment of a human Notch protein, said fragment consisting essentially of the intracellular domain of the protein, as encoded by a portion of plasmid hN3k as deposited with the ATCC and assigned accession number 68609.

### Claim 13

A substantially purified protein comprising: a fragment of a human Notch protein, said fragment consisting essentially of the intracellular domain of the protein, as encoded by a portion of plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 14

A substantially purified protein comprising: a fragment of a human Notch protein, said fragment consisting essentially of the region containing the cdc10 repeats of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 15

A substantially purified protein comprising: a fragment of a human Notch protein, said fragment consisting essentially of the region containing the cdc10 repeats, as encoded by a portion of plasmid hN3k as deposited with the ATCC and assigned accession number 68611.

### Claim 16

A substantially purified protein comprising: a fragment of a human Notch protein, said fragment consisting essentially of the region containing the cdc10 repeats, as encoded by a portion of plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 17

A substantially purified protein comprising a region of a human Notch protein containing the epidermal growth factor-like repeats of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 18

A substantially purified fragment of a human Notch protein substantially lacking the epidermal growth factor-like repeats of the protein, which fragment is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 19

A substantially purified fragment of a human Notch protein lacking a portion of the epidermal growth factor-like repeats of the protein, which fragment is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 20

A substantially purified protein comprising an amino acid sequence encoded by at least 121 nucleotides of the human cDNA sequence contained in plasmid hN3k as deposited with the ATCC and assigned accession number 68609.

### Claim 21

A substantially purified protein comprising an amino acid sequence encoded by at least 121 nucleotides of the human cDNA sequence contained in plasmid hN4k as deposited with the ATCC and assigned accession number 68610.

### Claim 22

A substantially purified protein comprising an amino acid sequence encoded by at least 121 nucleotides of the human cDNA sequence contained in plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 23

A substantially purified fragment of a human Notch protein consisting essentially of the intracellular domain of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 24

A substantially purified fragment of a human Notch protein consisting essentially of the extracellular domain of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 25

A substantially purified fragment of a human Notch protein consisting essentially of the extracellular and transmembrane domains of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 26 (depends on 18)

A chimeric protein comprising the fragment of claim 18 joined to a heterologous protein sequence.

### Claim 27 (depends on 19)

A chimeric protein comprising the fragment of claim 19 joined to a heterologous protein sequence.

### Claim 28

A substantially purified protein comprising a fragment of the Notch protein sequence encoded by the human cDNA sequence contained in plasmid hN3k as deposited with the ATCC and assigned accession number 68609, which fragment is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 29

A substantially purified protein comprising a fragment of the Notch protein sequence encoded by the human cDNA sequence contained in plasmid hN4k as deposited with the ATCC and assigned accession number 68610, which fragment is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 30

A substantially purified protein comprising a fragment of the Notch protein sequence encoded by the human cDNA sequence contained in plasmid hN5k as deposited with the ATCC and assigned accession number 68611, which fragment is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 31

A substantially purified protein comprising the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32).

### Claim 32

A substantially purified protein comprising the amino acid sequence depicted in FIG. 24 (SEQ ID NO:34).

### Claim 33

A substantially purified protein comprising the Notch amino acid sequence encoded by the human Notch DNA sequence contained in plasmid hN3k as deposited with the ATCC and assigned accession number 68609.

### Claim 34

A substantially purified protein comprising the Notch amino acid sequence encoded by the human Notch DNA sequence contained in plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 35

A substantially pure fragment of a Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 7-9.

### Claim 36

A substantially pure fragment of a Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal factor-like repeats 17-26.

### Claim 37

A substantially pure fragment of a Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 7-9 and 17-30.

### Claim 38

A substantially pure fragment of a Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 1-10 and 13-36.

### Claim 39

A substantially pure fragment of a Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment consists essentially of the portion of the Notch protein with the greatest homology over a sequence of at least 77 amino acids to the epidermal growth factor-like repeats 11 and 12 of the Drosophila Notch sequence as shown in FIG. 8 (SEQ ID NO:1).

### Claim 40 (depends on 35, 36, 37, 38)

The fragment of claim 35, 36, 37 or 38 in which the Notch protein is a Drosophila Notch protein.

### Claim 41 (depends on 35, 36, 37, 38)

The fragment of claim 35, 36, 37 or 38 in which the Notch protein is a Xenopus Notch protein.

### Claim 42 (depends on 35, 36, 37, 38)

A chimeric protein comprising the fragment of claim 35, 36, 37 or 38 joined to a heterologous protein sequence.

### Claim 43

A substantially purified protein comprising the portion of a human Notch protein with the greatest homology over a sequence of at least 77 amino acids, to the epidermal growth factor-like repeats 11 and 12 of the Drosophila Notch sequence as shown in FIG. 8 (SEQ ID NO:1), in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 44 (depends on 31, 32)

A substantially purified derivative of the protein of claim 31 or 32 having an insertion, substitution, or deletion relative to the protein, which derivative is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell.

### Claim 45

A substantially purified protein comprising: a fragment of a human Notch protein consisting of at least 77 amino acids, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 46

A substantially purified protein comprising: a fragment of a human Notch protein consisting essentially of the extracellular domain of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 47

A substantially purified protein comprising: a fragment of a human Notch protein consisting essentially of the intracellular domain of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 48

A substantially purified protein comprising: a fragment of a human Notch protein consisting essentially of the extracellular and transmembrane domains of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 49

A substantially purified protein comprising: a fragment of a human Notch protein, said fragment consisting essentially of the region containing the cdc10 repeats of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid sequence hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 50

A substantially purified protein comprising a region of a human Notch protein containing the epidermal growth factor-like repeats of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 51

A substantially purified protein comprising a region of a human Notch protein containing the Notch/lin-12 repeats of the protein, in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 52

A substantially purified protein comprising a region of a human Notch protein containing the Notch/lin-12 repeats of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 53

A substantially purified fragment of a human Notch protein substantially lacking the epidermal growth factor-like repeats of the protein, which fragment is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 54

A substantially purified fragment of a human Notch protein lacking a portion of the epidermal growth factor-like repeats of the protein, which fragment is able to be bound by an antibody to a human Notch protein but not to a Notch protein of another species, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid sequence hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 55

A substantially purified fragment of a human Notch protein consisting essentially of the intracellular domain of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 56

A substantially purified fragment of a human Notch protein consisting essentially of the extracellular domain of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 57

A substantially purified fragment of a human Notch protein consisting essentially of the extracellular and transmembrane domains of the protein, in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 58

A substantially purified fragment of a protein, said protein comprising the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32), which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell.

### Claim 59

A substantially purified fragment of a protein, said protein comprising the amino acid sequence depicted in FIG. 24 (SEQ ID NO:34), which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell.

### Claim 60

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 7-9, and in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 61

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 7-9, and in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 62

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 17-26, and in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 63

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 17-26, and in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 64

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 7-9 and 17-30, and in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 65

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 7-9 and 17-30, and in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 66

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 1-10 and 13-36, and in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO:34).

### Claim 67

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment does not contain epidermal growth factor-like repeats 1-10 and 13-36, and in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 68

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment consists essentially of the portion of the Notch protein with the greatest homology over a sequence of at least 77 amino acids to the epidermal growth factor-like repeats 11 and 12 of the Drosophila Notch sequence as shown in FIG. 8 (SEQ ID NO:1), and in which the human Notch protein comprises the amino acid sequence depicted in FIG. 23 (SEQ ID NO:32) or in FIG. 24 (SEQ ID NO: 34).

### Claim 69

A substantially purified fragment of a human Notch protein, which fragment is characterized by the ability in vitro, when expressed on the surface of a first cell to bind to a Delta protein expressed on the surface of a second cell, in which the fragment consists essentially of the portion of the Notch protein with the greatest homology over a sequence of at least 77 amino acids to the epidermal growth factor-like repeats 11 and 12 of the Drosophila Notch sequence as shown in FIG. 8 (SEQ ID NO:1), and in which the human Notch protein (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a first nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 70 (depends on 60, 62, 64, 66)

A chimeric protein comprising the fragment of claim 60, 62, 64 or 66 joined to a heterologous protein sequence.

### Claim 71 (depends on 31, 32)

A substantially purified fragment of the protein of claim 31 or 32, which fragment is characterized by the ability to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 72 (depends on 58, 59, 69)

A chimetic protein comprising the fragment of claim 58, 59 or 69, joined to a heterologous protein sequence.

### Claim 73 (depends on 31, 32)

A substantially purified derivative of the protein of claim 31 or 32 having an insertion, substitution or deletion relative to the protein, which derivative is characterized by the ability to be bound by an antibody to a human Notch protein but not to a Notch protein of another species.

### Claim 74 (depends on 73)

A chimeric protein comprising the derivative of claim 73 joined to a heterologous protein sequence.

### Claim 75

A substantially purified human protein that (a) is able to be bound by an antibody to a Notch protein, and (b) is encoded by a nucleic acid hybridizable to a second nucleic acid having the human Notch nucleotide sequence contained in (i) plasmid hN3k as deposited with the ATCC and assigned accession number 68609, or (ii) plasmid hN5k as deposited with the ATCC and assigned accession number 68611.

### Claim 76 (depends on 35)

A substantially purified derivative of the fragment of claim 35 having an insertion, substitution, or deletion relative to the fragment, which is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell, which derivative does not contain epidermal growth factor-like repeats 7-9 of the Notch protein.

### Claim 77 (depends on 36)

A substantially purified derivative of the fragment of claim 36 having an insertion, substitution, or deletion relative to the fragment, which is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell, which derivative does not contain epidermal growth factor-like repeats 17-26 of the Notch protein.

### Claim 78 (depends on 37)

A substantially purified derivative of tim fragment of claim 37 having an insertion, substitution, or deletion relative to the fragment, which is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell, which derivative does not contain epidermal growth factor-like repeats 7-9 and 17-30 of the Notch protein.

### Claim 79 (depends on 38)

A substantially purified derivative of the fragment of claim 38 having an insertion, substitution, or deflection relative to the fragment, which is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell, which derivative does not contain epidermal growth factor-like repeats 1-10 and 13-36 of the Notch protein.

### Claim 80 (depends on 60)

A substantially purified derivative of the fragment of claim 60 having an insertion, substitution, or deletion relative to the fragment, which derivative is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell, which derivative does not contain epidermal growth factor-like repeats 7-9 of the Notch protein.

### Claim 81 (depends on 62)

A substantially purified derivative of the fragment of claim 62 having an insertion, substitution, or deletion relative to the fragment, which derivative is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell, which derivative does not contain epidermal growth factor-like repeats 17-26 of the Notch protein.

### Claim 82 (depends on 64)

A substantially purified derivative of the fragment of claim 64 having an insertion, substitution, or deletion relative to the fragment, which derivative is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell, which derivative does not contain epidermal growth factor-like repeats 7-9 and 17-30 of the Notch protein.

### Claim 83 (depends on 66)

A substantially purified derivative of the fragment of claim 66 having an insertion, substitution, or deletion relative to the fragment, which derivative is characterized by the ability in vitro, when expressed on the surface of a first cell, to bind to a Delta protein expressed on the surface of a second cell which derivative does not contain epidermal growth factor-like repeats 1-10 and 13-36 of the Notch protein.