# US 2019/0169283 A1 · Anti-cxcr5 antibodies and compositions and uses thereof

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- Number: US 2019/0169283 A1
- Document ID: US20190169283A1
- Status: Published application
- Kind: A1
- Application number: 16/202,749
- Filed: 2018-11-28
- Published: 2019-06-06
- Earliest claimed: 2017-12-01
- Assignees: Pfizer Inc.; The Regents of the University of California
- Applicants: Pfizer Inc.; The Regents of the University of California
- Inventors: Rachel Groth; William Brian Snyder; Xianjun Cao; Robert Joseph Dunn; Joseph Dal Porto; Michael Karin
- Web page: https://patentgrep.com/patent/US20190169283A1

## CPC

- C07K 16/24: against cytokines, lymphokines or interferons
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C07K 16/40: against enzymes
- C12N 15/62: DNA sequences coding for fusion proteins
- A61K 2039/54: {characterised by the route of administration}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/30: characterized by aspects of specificity or valency
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- 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)
- 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 provides antibodies, and antigen-binding fragments thereof, that specifically bind to CXCR5. The antibodies can be afucosylated and exhibit increased ADCC compared with the otherwise identical fucosylated antibodies. The invention includes uses, and associated methods of using the antibodies.

## Claims

### Claim 1

An isolated antibody, or antigen-binding fragment thereof, that specifically binds C—X—C-chemokine receptor 5 (CXCR5),wherein the antibody, or antigen-binding fragment thereof, is at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of:
a) an antibody, or antigen-binding fragment thereof, comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2; a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3; a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4; a CDR-H1 comprising the amino acid sequence of SEQ ID NO:7; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:8; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:9;
b) an antibody, or antigen-binding fragment thereof, comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2; a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3; a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4; a CDR-H1 comprising the amino acid sequence of SEQ ID NO:7; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:8; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:11;
c) an antibody, or antigen-binding fragment thereof, comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:14; a CDR-L2 comprising the amino acid sequence of SEQ ID NO:15; a CDR-L3 comprising the amino acid sequence of SEQ ID NO:16; a CDR-H1 comprising the amino acid sequence of SEQ ID NO:19; a CDR-H2 comprising the amino acid sequence of SEQ ID NO:20; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:21;
d) an antibody, or antigen-binding fragment thereof, comprising a VL comprising an amino acid sequence encoded by the insert of the plasmid deposited with the ATCC and having Accession Number PTA-124324 and a VH comprising an amino acid sequence encoded by the insert of the plasmid deposited with the ATCC having Accession Number PTA-124323;
e) an antibody, or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of SEQ ID NO:1, and a VH comprising the amino acid sequence of SEQ ID NO:6;
f) an antibody, or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of SEQ ID NO:13, and a VH comprising the amino acid sequence of SEQ ID NO:18;
g) an antibody, or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of SEQ ID NO:47, and a VH comprising the amino acid sequence of SEQ ID NO:52;
h) an antibody, or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of SEQ ID NO:5, and a VH comprising the amino acid sequence of SEQ ID NO:6;
i) an antibody, or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of SEQ ID NO:5, and a VH comprising the amino acid sequence of SEQ ID NO:10;
j) an antibody, or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of SEQ ID NO:13, and a VH comprising the amino acid sequence of SEQ ID NO:17;
k) an antibody, or antigen-binding fragment thereof, comprising a VL comprising the amino acid sequence of SEQ ID NO:1, and a VH comprising the amino acid sequence of SEQ ID NO:12;
l) an antibody, or antigen-binding fragment thereof, comprising a LC comprising the amino acid sequence of SEQ ID NO:22, and a HC comprising the amino acid sequence of SEQ ID NO:23;
m) an antibody, or antigen-binding fragment thereof, comprising a LC comprising the amino acid sequence of SEQ ID NO:24, and a HC comprising the amino acid sequence of SEQ ID NO:25;
n) an antibody, or antigen-binding fragment thereof, comprising a LC comprising the amino acid sequence of SEQ ID NO:26, and a HC comprising the amino acid sequence of SEQ ID NO:27;
o) an antibody, or antigen-binding fragment thereof, comprising a LC comprising the amino acid sequence of SEQ ID NO:28, and a HC comprising the amino acid sequence of SEQ ID NO:29;
p) an antibody, or antigen-binding fragment thereof, comprising a VL encoded by the nucleic acid sequence of SEQ ID NO:95, and a VH encoded by the nucleic acid sequence of SEQ ID NO:96; and
q) an antibody, or antigen-binding fragment thereof, comprising a LC encoded by the nucleic acid sequence of SEQ ID NO:97, and a HC encoded by the nucleic acid sequence of SEQ ID NO:98.

### Claim 2

An isolated antibody or antigen-binding fragment thereof, that specifically binds CXCR5, wherein the antibody, or antigen-binding fragment thereof, is at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of:
a) an antibody or antigen-binding fragment thereof that binds a hCXCR5 epitope comprising leucine at amino acid residue number 11 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind said epitope wherein said residue is not leucine;
b) an antibody or antigen-binding fragment thereof that binds a hCXCR5 epitope comprising leucine at amino acid residue number 11 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind said epitope wherein said residue is threonine;
c) an antibody or antigen-binding fragment thereof that binds a hCXCR5 epitope comprising aspartate at amino acid residue number 22 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind said epitope wherein said residue is not aspartate;
d) an antibody or antigen-binding fragment thereof that binds a hCXCR5 epitope comprising aspartate at amino acid residue number 22 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind said epitope wherein said residue is alanine;
e) an antibody or antigen-binding fragment thereof, that binds a hCXCR5 epitope comprising leucine at amino acid residue number 11 and aspartate at amino acid residue number 22, according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind said epitope wherein said leucine is substituted by threonine and/or said aspartate is substituted with alanine;
f) an antibody or antigen-binding fragment thereof that binds hCXCR5, or a fragment thereof, comprising leucine at amino acid residue number 11 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind hCXCR5, or a fragment thereof, wherein said residue is not leucine;
g) an antibody or antigen-binding fragment thereof that binds hCXCR5, or a fragment thereof, comprising leucine at amino acid residue number 11 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind hCXCR5, or a fragment thereof, wherein said residue is threonine;
h) an antibody or antigen-binding fragment thereof that binds hCXCR5, or a fragment thereof, comprising aspartate at amino acid residue number 22 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind hCXCR5, or a fragment thereof, wherein said residue is not aspartate;
i) an antibody or antigen-binding fragment thereof that binds hCXCR5, or a fragment thereof, comprising aspartate at amino acid residue number 22 according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind hCXCR5, or a fragment thereof, wherein said residue is alanine; and
j) an antibody or antigen-binding fragment thereof, that binds hCXCR5, or a fragment thereof, comprising leucine at amino acid residue number 11 and aspartate at amino acid residue number 22, according to the numbering of the amino acid sequence of SEQ ID NO:32, but does not bind hCXCR5, or a fragment thereof, wherein said leucine is substituted by threonine and/or said aspartate is substituted with alanine.

### Claim 3

An isolated antibody or antigen-binding fragment thereof, that specifically binds CXCR5, wherein the antibody, or antigen-binding fragment thereof, is at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of:
a) an antibody, or antigen-binding fragment thereof, that binds hCXCR5 expressed on human B cells with an apparent affinity of an EC50 of about 6.60 pM with a standard deviation of about plus or minus 2.33 pM;
b) an antibody, or antigen-binding fragment thereof, that binds hCXCR5 expressed on human circulating follicular T helper-like cells with an apparent affinity of an EC50 of about 5.89 pM with a standard deviation of about plus or minus 1.40 pM;
c) an antibody, or antigen-binding fragment thereof, that binds hCXCR5 expressed on human follicular T helper (Tfh) cells with an apparent affinity of an EC50 of about 10.6 pM;
d) an antibody, or antigen-binding fragment thereof, that binds cynoCXCR5 expressed on cynomolgus monkey B cells with an apparent affinity of an EC50 of about 1.32 pM;
e) an antibody, or antigen-binding fragment thereof, that binds cynoCXCR5 expressed on cynomolgus monkey Tfh-like cells with an apparent affinity of an EC50 of about 10.5 pM;
f) an antibody, or antigen-binding fragment thereof, that antagonizes CXCRS-CXCL13 signaling in a cAMP reporter assay with an EC50 of about 961 pM;
g) an antibody, or antigen-binding fragment thereof, that exhibits ADCC activity against human B cells expressing hCXCR5 with an EC50 of about 2.01 pM with a standard deviation of about plus or minus 2.28 pM;
h) an antibody, or antigen-binding fragment thereof, that exhibits ADCC activity against human Tfh-like cells expressing hCXCR5 with an EC50 of about 4.28 pM with a standard deviation of about plus or minus 2.88 pM;
i) an antibody, or antigen-binding fragment thereof, that exhibits ADCC activity against human Tfh cells expressing hCXCR5 with an EC50 of about 0.11 pM;
j) an antibody, or antigen-binding fragment thereof, that exhibits ADCC activity against cynomolgus monkey B cells expressing cynoCXCR5 with an EC50 of about 15.3 pM with a standard deviation of about plus or minus 11.7 pM;
k) an antibody, or antigen-binding fragment thereof, that binds hCXCR5 but does not detectably bind human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7, and XCR1;
l) an antibody, or antigen-binding fragment thereof, that inhibits binding of CXCR5 to CXCL13;
m) an antibody, or antigen-binding fragment thereof, that binds CXCR5+ human B cells with an apparent affinity of an EC50 of less than about 26 pM, but does not bind cells expressing CXCR5 mouse, rat or rabbit orthologs;
n) an antibody, or antigen-binding fragment thereof, that antagonizes CXCL13 inhibition of cAMP release triggered by forskolin;
o) an antibody, or antigen-binding fragment thereof, that triggers ADCC of CXCR5-expressing cells in human donor and cynomolgus PBMCs and human donor TMCs;
p) an antibody, or antigen-binding fragment thereof, that binds human CXCR5 but does not bind human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7, or XCR1;
q) an antibody, or antigen-binding fragment thereof, that depletes B cells in the peripheral blood;
r) an antibody, or antigen-binding fragment thereof, that depletes Tfh-like cells in the peripheral blood;
s) an antibody, or antigen-binding fragment thereof, that depletes bona fide Tfh cells in the spleen; and
t) an antibody, or antigen-binding fragment thereof, that impairs humoral immune memory response.

### Claim 4 (depends on 1)

The antibody, or antigen-binding fragment thereof, of claim 1, wherein said antibody, or antigen-binding fragment thereof, exhibits at least one of the following biological activities:
a) binds CXCR5+ human B cells with an apparent affinity of an EC50 of less than about 26 pM, but does not bind cells expressing CXCR5 mouse, rat or rabbit orthologs;
b) antagonizes CXCL13 inhibition of cAMP release triggered by forskolin;
c) triggers ADCC of CXCR5-expressing cells in human donor and cynomolgus PBMCs and human donor TMCs;
d) binds human CXCR5 but does not bind human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7, or XCR1;
e) depletes B cells in the peripheral blood;
f) depletes Tfh-like cells in the peripheral blood;
g) depletes bona fide Tfh cells in the spleen; and
h) impairs a humoral immune memory response.

### Claim 5 (depends on 1)

The antibody, or antigen-binding fragment thereof, of claim 1, wherein said antibody, or antigen-binding fragment thereof, is afucosylated.

### Claim 6 (depends on 1)

An isolated nucleic acid encoding the antibody, or antigen-binding fragment thereof, claim 1.

### Claim 7 (depends on 6)

The isolated nucleic acid of claim 6, wherein the isolated nucleic acid encoding encodes the VH, VL, or both, of an antibody or antigen-binding fragment thereof that specifically binds CXCR5, and wherein said nucleic acid comprises: the nucleic acid sequence of SEQ ID NO:95, the nucleic acid sequence of SEQ ID NO:96, or both.

### Claim 8 (depends on 6)

The isolated nucleic acid of claim 6, wherein the isolated nucleic acid encodes the heavy chain, light chain, or both, of an antibody or antigen-binding fragment thereof that specifically binds CXCR5, and wherein said nucleic acid comprises: the nucleic acid sequence of SEQ ID NO:97, the nucleic acid sequence of SEQ ID NO:98, or both.

### Claim 9 (depends on 6)

The isolated nucleic acid of claim 6, wherein the isolated nucleic acid encodes the VH, VL, or both, of an antibody or antigen-binding fragment thereof that specifically binds CXCR5, and wherein said nucleic acid comprises the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the Accession Number PTA-124323, the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having the Accession Number PTA-124324, or both.

### Claim 10 (depends on 6)

A vector comprising the nucleic acid of claim 6.

### Claim 11 (depends on 10)

A host cell comprising the vector of claim 10.

### Claim 12 (depends on 11)

The host cell of claim 11, wherein said host cell is a mammalian cell selected from the group consisting of a CHO cell, a COS cell, a HEK-293 cell, an NSO cell, a PER.C6® cell, and an Sp2.0 cell.

### Claim 13 (depends on 12)

The host cell of claim 12, wherein said cell lacks a functional alpha-1,6-fucosyltransferase (FUT8).

### Claim 14 (depends on 13)

The host cell of claim 13, wherein said cell is a Potelligent® CHOK1SV cell or a Lec13 CHO cell.

### Claim 15 (depends on 14)

A method of making an antibody, or antigen-binding fragment thereof, comprising culturing the Potelligent® CHOK1SV cell of claim 14, under a condition wherein said antibody, or antigen-binding fragment thereof is expressed by said host cell and is afucosylated.

### Claim 16 (depends on 15)

The method of claim 15, further comprising isolating said antibody, or antigen-binding fragment thereof.

### Claim 17 (depends on 15)

The afucosylated antibody, or antigen-binding fragment thereof, made by the method of claim 15, wherein said antibody, or antigen-binding fragment thereof, exhibits enhanced ADCC activity when compared to an otherwise identical antibody, or antigen-binding fragment thereof, that is fucosylated.

### Claim 18 (depends on 1)

A pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, of claim 1, and a pharmaceutically acceptable carrier or excipient.

### Claim at position 19

19-20. (canceled)

### Claim 21 (depends on 18)

A method for treating an immune disease, disorder or condition mediated by CXCR5 in a human subject in need thereof, said method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 18, wherein said disease, disorder or condition is rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).

### Claim at position 21

22-23. (canceled)

### Claim 24 (depends on 1)

A method of detecting CXCR5 in a sample, tissue, or cell using the antibody, or antigen-binding fragment thereof, of claim 1, comprising contacting the sample, tissue or cell with the antibody, or antigen-binding fragment thereof, and detecting the antibody, or antigen-binding fragment thereof.

### Claim 25 (depends on 1)

A method of reducing a biological activity of CXCR5 in a subject in need thereof, said method comprising administering a therapeutically effective amount of the antibody, or antigen binding fragment thereof, of claim 1.

### Claim 26 (depends on 25)

The method of claim 25, wherein the antibody, or antigen-binding fragment thereof, mediates depletion of at least one cell expressing CXCR5 selected from the group consisting of a Tfh cell in the spleen, a B cell in peripheral blood, and a Tfh-like cell in peripheral blood.

### Claim 27 (depends on 1)

A method of inhibiting a humoral immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, of claim 1.

### Claim 28 (depends on 27)

The method of claim 27, wherein the antibody, or antigen-binding fragment thereof, mediates depletion of at least one cell expressing CXCR5 selected from the group consisting of a Tfh cell in the spleen, a B cell in peripheral blood, and a Tfh-like cell in peripheral blood.

### Claim 29 (depends on 1)

The isolated antibody, or antigen-binding fragment thereof, of claim 1, comprising a VL comprising the amino acid sequence of SEQ ID NO:1, and a VH comprising the amino acid sequence of SEQ ID NO:6.

### Claim 30 (depends on 1)

The isolated antibody, or antigen-binding fragment thereof, of claim 1, comprising a LC comprising the amino acid sequence of SEQ ID NO:28, and a HC comprising the amino acid sequence of SEQ ID NO:29.

### Claim 31

An isolated antibody that specifically binds CXCR5 comprising a LC consisting of the amino acid sequence of SEQ ID NO:28, and a HC consisting of the amino acid sequence of SEQ ID NO:29.

### Claim 32 (depends on 31)

The isolated antibody of claim 31, wherein the antibody is afucosylated.

### Claim 33

An isolated antibody that specifically binds CXCR5 comprising:
an antibody VL region comprising the first, second and third CDRs from the VL region comprising the amino acid sequence of SEQ ID NO:1; and
an antibody VH region comprising the first, second and third CDRs from the VH region comprising the amino acid sequence of SEQ ID NO:6.

### Claim 34 (depends on 33)

The antibody of claim 33, wherein the antibody is afucosylated.

### Claim 35

An isolated antibody that specifically binds CXCR5 comprising:
a) an antibody VL region comprising the first, second and third CDRs from the VL region comprising the amino acid sequence of SEQ ID NO:1;
an antibody VH region comprising the first, second and third CDRs from the VH region comprising the amino acid sequence of SEQ ID NO:6;
an antibody constant light (CL) region comprising the amino acid sequence of SEQ ID NO:30; and
an antibody constant heavy (CH) region comprising the amino acid sequence of SEQ ID NO:31; or
b) an antibody VL region at least 95% identical to the amino acid sequence of SEQ ID NO: 1; and an antibody VH region at least 95% identical to the amino acid sequence of SEQ ID NO:6.

### Claim 36 (depends on 35)

The antibody of claim 35, wherein the antibody is afucosylated.