# US 10,865,249 B2 · Antigen-binding polypeptides directed against CD38

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- Number: US 10,865,249 B2
- Document ID: US10865249B2
- Status: Granted
- Kind: B2
- Application number: 15/774,919
- Filed: 2016-11-10
- Published: 2019-09-12
- Granted: 2020-12-15
- Earliest claimed: 2015-11-10
- Assignees: University Medical Center Hamburg-Eppendorf
- Applicants: University Medical Center Hamburg-Eppendorf
- Inventors: Friedrich Nolte; Peter Bannas; Kerstin Schütze; William Fumey; Levin Schriewer; Stephan Menzel; Catelijne Stortelers
- Web page: https://patentgrep.com/patent/US10865249B2

## CPC

- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/06: Antiasthmatics
- A61P 13/02: of urine or of the urinary tract, e.g. urine acidifiers
- A61P 13/12: of the kidneys
- A61P 17/00: Drugs for dermatological disorders
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 15/86: Viral vectors
- C12Q 1/25: involving enzymes not classifiable in groups C12Q1/26 {- C12Q1/66}
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- A61K 2039/505: {comprising antibodies}
- C07K 2317/22: from camelids, e.g. camel, llama or dromedary
- C07K 2317/31: multispecific
- C07K 2317/64: comprising a combination of variable region and constant region components
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/734: Complement-dependent cytotoxicity [CDC]
- C07K 2317/75: Agonist effect on antigen
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)

## Abstract

The invention relates to polypeptides specifically binding to CD38 and are therefore suitable for the diagnosis and for the therapeutic and prophylactic treatment of diseases which are characterized by increased CD38 expression. Conjugates and pharmaceutical compositions comprising the polypeptides are disclosed as well. In addition, the invention relates to the use of such polypeptides in methods for the detection of CD38 and/or CD38-expressing cells in a biological sample. A process for the purification and concentration of CD38 and/or CD38-expressing cells in which the antigen-binding polypeptides are used are also described.

## Claims

### Claim 1

A polypeptide construct comprising:
a first immunoglobulin single variable domain (ISVD) that specifically binds to CD38 with an EC50 value of less than 200 nM, and
a second ISVD that specifically binds to CD38 with an EC50 value of less than 200 nM,
wherein said first ISVD binds a first epitope of CD38 and said second ISVD binds a second epitope of CD38, wherein said first epitope is different from said second epitope,
the polypeptide construct further comprising a CH2 and a CH3 constant domain and optionally the polypeptide construct comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers,
wherein said first ISVD and said second ISVD each comprises 3 complementarity determining regions (CDRs), in which:
(i) CDR1 is chosen from the group consisting of SEQ ID NOs: 117-174;
(ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 233-290; and
(iii) CDR3 is chosen from the group consisting of SEQ ID NOs: 349-406.

### Claim 2 (depends on 1)

The polypeptide construct according to claim 1, wherein said first or second ISVD is chosen from the group consisting of
ISVDs represented by SEQ ID NOs: 1 to 58.

### Claim 3 (depends on 1)

The polypeptide construct according to claim 1, wherein each set of 3 CDRs of said first ISVD and said second ISVD is selected from the group consisting of:
CDR1 is SEQ ID NO: 117, CDR2 is SEQ ID NO: 233 and CDR3 is SEQ ID NO: 349;
CDR1 is SEQ ID NO: 118, CDR2 is SEQ ID NO: 234 and CDR3 is SEQ ID NO: 350;
CDR1 is SEQ ID NO: 119, CDR2 is SEQ ID NO: 235 and CDR3 is SEQ ID NO: 351;
CDR1 is SEQ ID NO: 120, CDR2 is SEQ ID NO: 236 and CDR3 is SEQ ID NO: 352;
CDR1 is SEQ ID NO: 121, CDR2 is SEQ ID NO: 237 and CDR3 is SEQ ID NO: 353;
CDR1 is SEQ ID NO: 122, CDR2 is SEQ ID NO: 238 and CDR3 is SEQ ID NO: 354;
CDR1 is SEQ ID NO: 123, CDR2 is SEQ ID NO: 239 and CDR3 is SEQ ID NO: 355;
CDR1 is SEQ ID NO: 124, CDR2 is SEQ ID NO: 240 and CDR3 is SEQ ID NO: 356;
CDR1 is SEQ ID NO: 125, CDR2 is SEQ ID NO: 241 and CDR3 is SEQ ID NO: 357;
CDR1 is SEQ ID NO: 126, CDR2 is SEQ ID NO: 242 and CDR3 is SEQ ID NO: 358;
CDR1 is SEQ ID NO: 127, CDR2 is SEQ ID NO: 243 and CDR3 is SEQ ID NO: 359;
CDR1 is SEQ ID NO: 128, CDR2 is SEQ ID NO: 244 and CDR3 is SEQ ID NO: 360;
CDR1 is SEQ ID NO: 129, CDR2 is SEQ ID NO: 245 and CDR3 is SEQ ID NO: 361;
CDR1 is SEQ ID NO: 130, CDR2 is SEQ ID NO: 246 and CDR3 is SEQ ID NO: 362;
CDR1 is SEQ ID NO: 131, CDR2 is SEQ ID NO: 247 and CDR3 is SEQ ID NO: 363;
CDR1 is SEQ ID NO: 132, CDR2 is SEQ ID NO: 248 and CDR3 is SEQ ID NO: 364;
CDR1 is SEQ ID NO: 133, CDR2 is SEQ ID NO: 249 and CDR3 is SEQ ID NO: 365;
CDR1 is SEQ ID NO: 134, CDR2 is SEQ ID NO: 250 and CDR3 is SEQ ID NO: 366;
CDR1 is SEQ ID NO: 135, CDR2 is SEQ ID NO: 251 and CDR3 is SEQ ID NO: 367;
CDR1 is SEQ ID NO: 136, CDR2 is SEQ ID NO: 252 and CDR3 is SEQ ID NO: 368;
CDR1 is SEQ ID NO: 137, CDR2 is SEQ ID NO: 253 and CDR3 is SEQ ID NO: 369;
CDR1 is SEQ ID NO: 138, CDR2 is SEQ ID NO: 254 and CDR3 is SEQ ID NO: 370;
CDR1 is SEQ ID NO: 139, CDR2 is SEQ ID NO: 255 and CDR3 is SEQ ID NO: 371;
CDR1 is SEQ ID NO: 140, CDR2 is SEQ ID NO: 256 and CDR3 is SEQ ID NO: 372;
CDR1 is SEQ ID NO: 141, CDR2 is SEQ ID NO: 257 and CDR3 is SEQ ID NO: 373;
CDR1 is SEQ ID NO: 142, CDR2 is SEQ ID NO: 258 and CDR3 is SEQ ID NO: 374;
CDR1 is SEQ ID NO: 143, CDR2 is SEQ ID NO: 259 and CDR3 is SEQ ID NO: 375;
CDR1 is SEQ ID NO: 144, CDR2 is SEQ ID NO: 260 and CDR3 is SEQ ID NO: 376;
CDR1 is SEQ ID NO: 145, CDR2 is SEQ ID NO: 261 and CDR3 is SEQ ID NO: 377;
CDR1 is SEQ ID NO: 146, CDR2 is SEQ ID NO: 262 and CDR3 is SEQ ID NO: 378;
CDR1 is SEQ ID NO: 147, CDR2 is SEQ ID NO: 263 and CDR3 is SEQ ID NO: 379;
CDR1 is SEQ ID NO: 148, CDR2 is SEQ ID NO: 264 and CDR3 is SEQ ID NO: 380;
CDR1 is SEQ ID NO: 149, CDR2 is SEQ ID NO: 265 and CDR3 is SEQ ID NO: 381;
CDR1 is SEQ ID NO: 150, CDR2 is SEQ ID NO: 266 and CDR3 is SEQ ID NO: 382;
CDR1 is SEQ ID NO: 151, CDR2 is SEQ ID NO: 267 and CDR3 is SEQ ID NO: 383;
CDR1 is SEQ ID NO: 152, CDR2 is SEQ ID NO: 268 and CDR3 is SEQ ID NO: 384;
CDR1 is SEQ ID NO: 153, CDR2 is SEQ ID NO: 269 and CDR3 is SEQ ID NO: 385;
CDR1 is SEQ ID NO: 154, CDR2 is SEQ ID NO: 270 and CDR3 is SEQ ID NO: 386;
CDR1 is SEQ ID NO: 155, CDR2 is SEQ ID NO: 271 and CDR3 is SEQ ID NO: 387;
CDR1 is SEQ ID NO: 156, CDR2 is SEQ ID NO: 272 and CDR3 is SEQ ID NO: 388;
CDR1 is SEQ ID NO: 157, CDR2 is SEQ ID NO: 273 and CDR3 is SEQ ID NO: 389;
CDR1 is SEQ ID NO: 158, CDR2 is SEQ ID NO: 274 and CDR3 is SEQ ID NO: 390;
CDR1 is SEQ ID NO: 159, CDR2 is SEQ ID NO: 275 and CDR3 is SEQ ID NO: 391;
CDR1 is SEQ ID NO: 160, CDR2 is SEQ ID NO: 276 and CDR3 is SEQ ID NO: 392;
CDR1 is SEQ ID NO: 161, CDR2 is SEQ ID NO: 277 and CDR3 is SEQ ID NO: 393;
CDR1 is SEQ ID NO: 162, CDR2 is SEQ ID NO: 278 and CDR3 is SEQ ID NO: 394;
CDR1 is SEQ ID NO: 163, CDR2 is SEQ ID NO: 279 and CDR3 is SEQ ID NO: 395;
CDR1 is SEQ ID NO: 164, CDR2 is SEQ ID NO: 280 and CDR3 is SEQ ID NO: 396;
CDR1 is SEQ ID NO: 165, CDR2 is SEQ ID NO: 281 and CDR3 is SEQ ID NO: 397;
CDR1 is SEQ ID NO: 166, CDR2 is SEQ ID NO: 282 and CDR3 is SEQ ID NO: 398;
CDR1 is SEQ ID NO: 167, CDR2 is SEQ ID NO: 283 and CDR3 is SEQ ID NO: 399;
CDR1 is SEQ ID NO: 168, CDR2 is SEQ ID NO: 284 and CDR3 is SEQ ID NO: 400;
CDR1 is SEQ ID NO: 169, CDR2 is SEQ ID NO: 285 and CDR3 is SEQ ID NO: 401;
CDR1 is SEQ ID NO: 170, CDR2 is SEQ ID NO: 286 and CDR3 is SEQ ID NO: 402;
CDR1 is SEQ ID NO: 171, CDR2 is SEQ ID NO: 287 and CDR3 is SEQ ID NO: 403;
CDR1 is SEQ ID NO: 172, CDR2 is SEQ ID NO: 288 and CDR3 is SEQ ID NO: 404;
CDR1 is SEQ ID NO: 173, CDR2 is SEQ ID NO: 289 and CDR3 is SEQ ID NO: 405; and
CDR1 is SEQ ID NO: 174, CDR2 is SEQ ID NO: 290 and CDR3 is SEQ ID NO: 406.

### Claim 4 (depends on 1)

The polypeptide construct according to claim 1,
wherein said first ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and said 3 CDRs, in which:
(i) CDR1 is chosen from the group consisting of SEQ ID NOs: 131, 132, 134, 140, 144, 146, 150, 151, 152, 153, 155, and 158, and
(ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 247, 248, 250, 256, 260, 262, 266, 267, 268, 269, 271, and 274, and
(iii) CDR3 is chosen from the group consisting of SEQ ID NOs: 363, 364, 366, 372, 376, 378, 382, 383, 384, 385, 387, and 390; and
said second ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and said 3 CDRs, in which:
(i) CDR1 is chosen from the group consisting of SEQ ID NOs: 129, 163, 164, 165, and 166, and
(ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 245, 279, 280, 281, and 282, and
(iii) CDR3 is chosen from the group consisting of SEQ ID NOs: 361, 395, 396, 397, and 398; or
wherein said first ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and said 3 CDRs, in which:
(i) CDR1 is chosen from the group consisting of SEQ ID NOs: 131, 132, 134, 140, 144, 146, 150, 151, 152, 153, 155, and 158, and
(ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 247, 248, 250, 256, 260, 262, 266, 267, 268, 269, 271, and 274, and
(iii) CDR3 is chosen from the group consisting of SEQ ID NOs: 363, 364, 366, 372, 376, 378, 382, 383, 384, 385, 387, and 390; and
said second ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and said 3 CDRs, in which:
(i) CDR1 is chosen from the group consisting of SEQ ID NOs: 117, 119, 120, 125, 126, 136, 159, 160, 161, 162, 167, 168, 169, 170, 171, 172, 173, and 174, and
(ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 233, 235, 236, 241, 242, 252, 275, 276, 277, 278, 283, 284, 285, 286, 287, 288, 289, and 290, and
(iii) CDR3 is chosen from the group consisting of SEQ ID NOs: 349, 351, 352, 357, 358, 368, 391, 392, 393, 394, 399, 400, 401, 402, 403, 404, 405, and 406; or
wherein said first ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and said 3 CDRs, in which:
(i) CDR1 is chosen from the group consisting of SEQ ID NOs: 129, 163, 164, 165, and 166, and
(ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 245, 279, 280, 281, and 282, and
(iii) CDR3 is chosen from the group consisting of SEQ ID NOs: 361, 395, 396, 397, and 398; and
said second ISVD essentially consists of 4 framework regions (FR1 to FR4, respectively) and said 3 CDRs, in which:
(i) CDR1 is chosen from the group consisting of SEQ ID NOs: 117, 119, 120, 125, 126, 136, 159, 160, 161, 162, 167, 168, 169, 170, 171, 172, 173, and 174, and
(ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 233, 235, 236, 241, 242, 252, 275, 276, 277, 278, 283, 284, 285, 286, 287, 288, 289, and 290, and
(iii) CDR3 is chosen from the group consisting of SEQ ID NOs: 349, 351, 352, 357, 358, 368, 391, 392, 393, 394, 399, 400, 401, 402, 403, 404, 405, and 406.

### Claim 5 (depends on 1)

The polypeptide construct according to claim 1,
wherein the EC50 in a FACS assay is 190 pM or less; and/or
wherein said polypeptide binds to CD38 with an IC50 of at most 100 nM as determined by a competition FACS; and/or
wherein said polypeptide binds to CD38 with an IC50 which is at least 10% better than the IC50 of a benchmark.

### Claim 6 (depends on 1)

A pharmaceutical composition comprising a polypeptide construct according to claim 1.

### Claim 7 (depends on 1)

The polypeptide construct according to claim 1,
wherein the EC50 in a FACS assay is less than 16 pM; and/or
wherein said polypeptide binds to CD38 with an IC50 of at most 2 nM as determined by a competition FACS; and/or
wherein said polypeptide binds to CD38 with an IC50 which is at least 50% better than the IC50 of a benchmark.

### Claim 8 (depends on 1)

The polypeptide construct according to claim 1, wherein said first and second ISVDs each specifically bind to human CD38 (SEQ ID NO: 465).

### Claim 9 (depends on 1)

The polypeptide construct according to claim 1, wherein said first epitope on CD38 does not overlap with said second epitope on CD38.

### Claim 10 (depends on 1)

A nucleic acid molecule that encodes a polypeptide construct according to claim 1.

### Claim 11 (depends on 10)

An expression vector comprising a nucleic acid molecule according to claim 10.

### Claim 12 (depends on 10)

A host cell comprising a nucleic acid molecule according to claim 10.

### Claim 13 (depends on 12)

A method for the recombinant production of a polypeptide, comprising (a) culturing the host cell of claim 12 under conditions which allow the expression of the nucleic acid molecule; and (b) isolating the polypeptide from the culture.

### Claim 14 (depends on 1)

A method of therapeutic treatment comprising administering, to a subject in need thereof, a pharmaceutically active amount of a polypeptide construct according to claim 1 for therapeutic treatment of a disease which is characterized by increased CD38 expression.

### Claim 15 (depends on 14)

The method of claim 14, wherein said disease which is characterized by increased CD38 expression is a hyperproliferative disease or an autoimmune disease.

### Claim 16 (depends on 1)

A method of therapeutic treatment comprising administering, to a subject in need thereof, a pharmaceutically active amount of a polypeptide construct according to claim 1 for therapeutic treatment of Burkitt's lymphoma, T-cell lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), multiple myeloma, chronic myelogenous leukemia (CIVIL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), CD38-expressing solid tumor, systemic lupus erythematosus (SLE), rheumatoid arthritis, Crohn's disease, ulcerative colitis, Hashimoto's thyroiditis, ankylosing spondylitis, multiple sclerosis, Graves' disease, Sjögren's syndrome, polymyositis, bullous pemphigoid, glomerulonephritis, vasculitis or asthma, Barraquer-Simons Syndrome, autoimmune heart disease, inflammatory bowel disease, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, ischemia-reperfusion injuries or rejection of transplanted organs.

### Claim 17

A method for determining competitor polypeptides competing with a polypeptide represented by SEQ ID NOs: 1-58, comprising
determining binding of said competitor polypeptide in the presence of polypeptide represented by SEQ ID NOs: 1-58 to CD38;
detecting a competitor polypeptide when the binding to CD38 of said competitor polypeptide is reduced by at least 10% in the presence of a polypeptide represented by SEQ ID NOs: 1-58, compared to the binding to CD38 of the competitor in the absence of the polypeptide represented by SEQ ID NOs: 1-58.