# US 2014/0154253 A1 · Bispecific Asymmetric Heterodimers Comprising Anti-CD3 Constructs

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- Number: US 2014/0154253 A1
- Document ID: US20140154253A1
- Status: Published application
- Kind: A1
- Application number: 13/941,449
- Filed: 2013-07-13
- Published: 2014-06-05
- Earliest claimed: 2012-07-13
- Assignees: Zymeworks Inc.
- Applicants: Zymeworks Inc.
- Inventors: Gordon Yiu Kon NG; Surjit Bhimarao DIXIT; Thomas SPRETER VON KREUDENSTEIN
- Web page: https://patentgrep.com/patent/US20140154253A1

## CPC

- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/12: Antivirals
- A61P 33/14: Ectoparasiticides, e.g. scabicides
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2887: {against CD20}
- C07K 16/32: against translation products of oncogenes
- C07K 2317/31: multispecific
- C07K 2317/35: Valency
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/528: CH4 domain
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/64: comprising a combination of variable region and constant region components
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/72: Increased effector function due to an Fc-modification
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/74: Inducing cell proliferation
- 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 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- 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

## Abstract

Disclosed herein are isolated multi-specific heteromultimer constructs that bind to CD3 expressed on T-cells and to an antigen expressed on B-cells. The multi-specific heteromultimer constructs are capable of bridging T- and B-cells and mediating killing of B-cells. The multi-specific heteromultimer constructs are based on a heterodimeric Fc scaffold or on a segmented albumin scaffold. Also disclosed herein are multi-specific heteromultimer constructs that bind to HER2 and HER3.

## Claims

### Claim 1

An isolated multispecific heteromultimer construct comprising:
a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell;
a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and an antigen binding polypeptide construct that binds to a target antigen on at least one B cell; wherein:
at least one of said CD3 binding polypeptide construct and said antigen binding polypeptide construct comprises a single chain Fv region;
said multispecific heteromultimer construct simultaneously engages said at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell; and
said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein:
said heterodimeric Fc is formed with stability at least comparable to a native homodimeric Fc, and
said heterodimeric Fc is formed with purity such that when said multispecific heteromultimer construct is expressed in a mammalian cell in an expression product, said expression product comprises at least about 70% of said multispecific heteromultimer, and less than 10% monomers or homodimers of said first or second polypeptide constructs.

### Claim 2 (depends on 1)

The isolated multispecific heteromultimer of claim 1, wherein said first or second polypeptide construct is devoid of at least one of immunoglobulin light chain, and immunoglobulin first constant (CH1) region.

### Claim 3 (depends on 1)

The isolated multispecific heteromultimer of claim 1, wherein the heterodimer Fc region comprises a variant CH2 domain or hinge comprising amino acid modifications that prevents functionally effective binding to all the Fcgamma receptors, and/or to complement proteins (C1q complex).

### Claim 4

(canceled)

### Claim 5 (depends on 1)

The isolated multispecific heteromultimer of claim 1, wherein the heterodimer Fc region comprises a variant CH2 domain or hinge comprising amino acid modifications that enhance binding to the FcγRIIb receptor.

### Claim 6

An isolated multispecific heteromultimer construct comprising:
a first polypeptide construct comprising a first heavy chain polypeptide and a CD3 binding polypeptide construct that binds to a CD3 complex on at least one CD3 expressing cell;
a second polypeptide construct comprising a second heavy chain polypeptide which is different from said first heavy chain polypeptide, and an antigen binding polypeptide construct that binds to a target antigen on at least one B cell; wherein:
at least one of said CD3 binding polypeptide construct and said antigen binding polypeptide construct optionally comprises a single chain Fv region;
said first and second heavy chain polypeptides form a heterodimeric Fc region comprising a variant immunoglobulin CH3 region comprising at least one amino acid mutation that promotes the formation of said heterodimeric Fc, wherein:
said heterodimeric Fc is formed with stability at least comparable to a native homodimeric Fc, and
said heterodimeric Fc is formed with purity such that when said multispecific heteromultimer construct is coexpressed from a mammalian cell in an expression product, said expression product comprises greater than 70% of said multispecific heteromultimer, and less than 10% monomers or homodimers of said first or second polypeptide constructs; and
said multispecific heteromultimer construct binds said at least one B cell with a valency greater than one, and said multispecific heteromultimer simultaneously engages said at least one B cell and said at least one CD3 expressing cell such that the CD3 expressing cell is activated, thereby inducing killing of the B cell.

### Claim 7

(canceled)

### Claim 8

(canceled)

### Claim 9 (depends on 1)

The isolated multispecific heteromultimer of claim 1 wherein the heterodimer Fc region comprises a variant CH2 domain comprising amino acid modifications to promote selective binding of a Fcgamma receptor.

### Claim 10

(canceled)

### Claim 11

(canceled)

### Claim 12 (depends on 1)

The isolated multispecific heteromultimer according to claim 1, wherein the variant CH3 domain has a melting temperature (Tm) of about 73° C. or greater.

### Claim 13

(canceled)

### Claim 14

(canceled)

### Claim 15 (depends on 1)

The isolated multispecific heteromultimer according to claim 1, wherein the heterodimer Fc region is formed with a purity of at least about 90% and/or the Tm is about 75° C.

### Claim 16 (depends on 1)

The isolated multispecific heteromultimer according to claim 1 wherein:
a. the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V, and the variant CH3 sequence of the second transporter polypeptide comprises the amino acid modifications T366L, K392M, and T394W;
b. the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V, and the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T366L, K392L, and T394W;
c. the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, T366L, K392M, and T394W;
d. the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, T366L, K392L, and T394W;
e. the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T366L, N390R, K392R, and T394W, and the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications L351Y, S400E, F405A, and Y407V; or
f. the variant CH3 sequence of the first heavy chain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392R, and T394W, and the variant CH3 sequence of the second heavy chain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A, and Y407V.

### Claim 17 (depends on 1)

The isolated multispecific heteromultimer of claim 1 wherein the heterodimer Fc is glycosylated, afucosylated, and/or aglycosylated.

### Claim 18

(canceled)

### Claim 19

(canceled)

### Claim 20 (depends on 1)

The isolated multispecific heteromultimer of claim 1, wherein said antigen binding polypeptide construct that binds to a target antigen on at least one B cell comprises at least one target antigen binding domain derived from an antibody, a fibronectin, an affibody, anticalin, cysteine knot protein, DARPin, avimer, Kunitz domain or variant or derivative thereof.

### Claim 21 (depends on 20)

The isolated multispecific heteromultimer of claim 20, wherein said antibody is a heavy chain antibody devoid of light chains.

### Claim 22 (depends on 1)

The isolated multispecific heteromultimer of claim 1, wherein said antigen binding polypeptide construct comprises at least one CD19 binding domain or at least one CD20 binding domain.

### Claim at position 23

-34. (canceled)

### Claim 35 (depends on 1)

The isolated multispecific heteromultimer of claim 1, wherein said at least one CD3 binding polypeptide construct comprises at least one CD3 binding domain derived from a CD3 specific antibody, a nanobody, fibronectin, affibody, anticalin, cysteine knot protein, DARPin, avimer, Kunitz domain or variant or derivative thereof.

### Claim 36 (depends on 35)

The isolated multispecific heteromultimer of claim 35, wherein said at least one CD3 binding domain comprises at least one amino acid modification that reduces immunogenicity as compared to a corresponding CD3 binding domain not comprising said modification, or at least one amino acid modification that increases its stability as measured by Tm, as compared to a corresponding CD3 binding domain not comprising said modification.

### Claim 37

(canceled)

### Claim 38 (depends on 36)

The isolated multispecific heteromultimer of claim 36, wherein said CD3 specific antibody is a heavy chain antibody devoid of light chains.

### Claim 39

(canceled)

### Claim 40 (depends on 1)

The isolated multispecific heteromultimer of claim 1 wherein both of said first and second polypeptide constructs comprise a single-chain Fv polypeptide.

### Claim 41 (depends on 1)

The isolated multispecific heteromultimer of claim 1 wherein at least one of said first and second polypeptide constructs further comprises a single-chain Fab polypeptide.

### Claim 42 (depends on 1)

The isolated multispecific heteromultimer of claim 1 wherein the CD3 expressing cell is a T-cell, and wherein said heteromultimer binds to the T-cell with sufficient affinity and decorates the T cell at sufficient capacity that induces the T-cell to display B cell killing activity when the T cell and the B cell are bridged.

### Claim 43

(canceled)

### Claim 44 (depends on 1)

The isolated multispecific heteromultimer of claim 1 where in the CD3 expressing cell is a human cell, or a non-human, mammalian cell.

### Claim 45

(canceled)

### Claim 46 (depends on 1)

The isolated multispecific heteromultimer of claim 1 where in the at least one CD3 binding polypeptide construct binds to CD3 constructs across multiple species.

### Claim 47 (depends on 1)

The isolated multispecific heteromultimer of claim 1 wherein the at least one B cell is associated with a cancer, or is an autoimmune reactive cell that is a lymphoid or myeloid cell.

### Claim 48 (depends on 47)

The isolated multispecific heteromultimer of claim 47 wherein the disease is a cancer selected from a carcinoma, a sarcoma, leukaemia, lymphoma and glioma.

### Claim at position 38

-51. (canceled)

### Claim 52 (depends on 1)

The isolated multispecific heteromultimer of claim 1 wherein said heteromultimer optionally comprises a linker between the CD3 binding polypeptide construct and said heterodimeric Fc and/or between the antigen binding polypeptide construct and said heterodimeric Fc.

### Claim 53 (depends on 52)

The isolated multispecific heteromultimer of claim 52, wherein said at least one linker is a polypeptide comprising from about 1 to about 100 amino acids.

### Claim 54 (depends on 1)

A set of expression vectors for expressing the multispecific heteromultimer claim 1, comprising at least a first DNA sequence encoding said first polypeptide construct and at least a second DNA sequence encoding said second polypeptide construct.

### Claim 55 (depends on 1)

A method of producing an expression product containing a multispecific heteromultimer of claim 1, in stable mammalian cells, the method comprising:
transfecting at least one mammalian cell with: at least a first DNA sequence encoding said first polypeptide construct and at least a second DNA sequence encoding said second polypeptide construct, such that said at least one first DNA sequence, said at least one second DNA sequence are transfected in said at least one mammalian cell in a pre-determined ratio to generate stable mammalian cells; culturing said stable mammalian cells to produce said expression product comprising said multispecific heteromultimer.

### Claim at position 43

-57. (canceled)

### Claim 58 (depends on 1)

A pharmaceutical composition comprising the multispecific heteromultimer of claim 1, and a suitable excipient.

### Claim 59 (depends on 1, 57)

A process for the production of a pharmaceutical composition of claim 57, said process comprising:
culturing a host cell under conditions allowing the expression of a heteromultimer of claim 1;
recovering the produced heteromultimer from the culture; and
producing the pharmaceutical composition.

### Claim 60

(canceled)

### Claim 61 (depends on 1)

A method of treating cancer in a mammal in need thereof, comprising administering to the mammal a composition comprising an effective amount of the heteromultimer of claim 1, optionally in combination with other pharmaceutically active molecules.

### Claim at position 48

-67. (canceled)

### Claim 68 (depends on 1)

A method of treating a cancer non-responsive to at least one of a CD19 lytic antibody, a CD20 lytic antibody and blinatumomab, in a mammal in need thereof, comprising administering to the mammal a composition comprising an effective amount of the heteromultimer of claim 1.

### Claim at position 50

-71. (canceled)

### Claim 72 (depends on 1)

A method of treating an autoimmune condition or an inflammatory condition in a mammal in need thereof, comprising administering to said mammal a composition comprising an effective amount of the heteromultimer of claim 1.

### Claim at position 52

-74. (canceled)

### Claim 75 (depends on 1)

A kit comprising a heteromultimer of claim 1, and instructions for use thereof.