EA043381B1 - ANTIBODIES TO FAM19A5 AND THEIR APPLICATION - Google Patents

Description

Link to the list of sequences is provided in electronic form

Contents of the list of sequences presented electronically in a text file

ASCII (name: 3763.005PC02_SeqListing_ST25.txt; size: 166,889 bytes and creation date:

June 2018), filed in the application for this invention, is incorporated by reference in its entirety.

Field of technology

The present invention provides antibodies that specifically bind to family member A5 with sequence similarity 19 (FAM19A5), compositions containing such antibodies, and a method of using such antibodies to prevent or treat disorders or diseases such as fibrosis and/or cancer (eg, tumor brain, such as glioblastoma) in the subject.

State of the art

FAM19A5 is a member of the TAFA protein subfamily, which consists of five highly homologous small proteins. Tang T.Y. et al., Genomics, 83(4):727–34 (2004). These proteins contain conserved cysteine residues in fixed positions and are distantly related to macrophage inflammatory protein 1 alpha (MIP-1 alpha), a member of the CC chemokine family. TAFA proteins are predominantly expressed in certain regions of the brain and spinal cord. These proteins are thought to be produced and secreted by mature neuronal stem cells during neurogenesis.

FAM19A5 is predominantly expressed in the vertebrate brain and FAM19A5 is believed to play an important role in the development, differentiation, formation of a complete central nervous system, and can be used to prevent or treat damage and/or disease of the central nervous system. US Patent Publication No. 2015/0118230.

In addition to regulating the nervous system, FAM19A5 may also play a role in regulating immune cells. Fibrosis is a common health problem, often occurring in various pathological processes, and is characterized by the infiltration of mononuclear immune cells that release cytokines that stimulate fibroblasts to change connective tissue. Accordingly, there is a need to develop antibodies that specifically bind to FAM19A5 and that are capable of modulating the activity of FAM19A5.

Disclosure of the Invention

Provided herein is an isolated antibody, or antigen-binding portion thereof, that specifically binds to the human family with sequence similarity member 19 (FAM19A5) protein A5 (anti-FAM19A5 antibody) and that competes with a reference antibody containing CDR1 for binding to the human FAM19A5 epitope. CDR2 and CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain, where:

(i) Heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 11, Heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 12,

The heavy chain CDR3 contains the amino acid sequence SEQ ID NO:13,

The light chain CDR1 contains the amino acid sequence SEQ ID NO:23,

Light chain CDR2 contains the amino acid sequence of SEQ ID NO: 24 and light chain CDR3 contains the amino acid sequence of SEQ ID NO: 25;

(ii) Heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 14, Heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 15,

The heavy chain CDR3 contains the amino acid sequence SEQ ID NO:16,

The light chain CDR1 contains the amino acid sequence SEQ ID NO:26,

Light chain CDR2 contains the amino acid sequence of SEQ ID NO: 27 and light chain CDR3 contains the amino acid sequence of SEQ ID NO: 28; or (iii) the heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 20, the heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 21,

The heavy chain CDR3 contains the amino acid sequence SEQ ID NO:22,

The light chain CDR1 contains the amino acid sequence SEQ ID NO:32,

Light chain CDR2 contains the amino acid sequence of SEQ ID NO: 33 and light chain CDR3 contains the amino acid sequence of SEQ ID NO: 34.

Also provided herein are an isolated antibody, or an antigen-binding portion thereof, that specifically binds to the human family with sequence similarity 19 (FAM19A5) member A5 protein (anti-FAM19A5 antibody), and that binds to the same epitope of human FAM19A5 as the reference antibody, containing CDR1, CDR2 and CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain, where:

(i) Heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 11, Heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 12,

The heavy chain CDR3 contains the amino acid sequence SEQ ID NO:13,

The light chain CDR1 contains the amino acid sequence SEQ ID NO:23,

The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 24 and

- 1 043381

The light chain CDR3 contains the amino acid sequence SEQ ID NO: 25;

(ii) Heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 14, Heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 15,

The heavy chain CDR3 contains the amino acid sequence SEQ ID NO:16,

The light chain CDR1 contains the amino acid sequence SEQ ID NO:26,

The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 27 and

The light chain CDR3 contains the amino acid sequence SEQ ID NO: 28; or (iii) the heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 20,

The heavy chain CDR2 contains the amino acid sequence SEQ ID NO:21,

The heavy chain CDR3 contains the amino acid sequence SEQ ID NO:22,

The light chain CDR1 contains the amino acid sequence SEQ ID NO:32,

The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 33 and

The light chain CDR3 contains the amino acid sequence SEQ ID NO: 34.

In some embodiments, the anti-FAM19A5 antibody of the present invention binds to at least the FAM19A5 epitope represented by SEQ ID NO: 6. In certain embodiments, the anti-FAM19A5 antibody binds to the FAM19A5 epitope represented by SEQ ID NO: 6 to one or more amino acids, respectively, amino acid residues (i) 46-51 (i.e. DSSQPR), (ii) 46, 50 and 52 (i.e. D-P-R) or (iii) 46, 47, 48 and 50 (i.e. .DSS-P). In some embodiments, an anti-FAM19A5 antibody binds to at least one FAM19A5 epitope, defined as SEQ ID NO: 9. In certain embodiments, an anti-FAM19A5 antibody binds to at least one FAM19A5 epitope, defined as EP2, EP4, and/or EP8, wherein EP2 contains the amino acids DSSQP (SEQ ID NO: 66), where EP4 contains the amino acids ARCACRK (SEQ ID NO: 68) and where EP8 contains the amino acids TCTQPGGR (SEQ ID NO: 72).

In some embodiments, an anti-FAM19A5 antibody described herein comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein:

(i) the heavy chain CDR1 contains SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 20;

(ii) the heavy chain CDR2 contains SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 21;

(iii) the heavy chain CDR3 contains SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 22;

(iv) the light chain CDR1 contains SEQ ID NO: 23, SEQ ID NO: 26 or SEQ ID NO: 32;

(v) the light chain CDR2 contains SEQ ID NO: 24, SEQ ID NO: 27 or SEQ ID NO: 33; and/or (vi) the light chain CDR3 comprises SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO: 34.

In some embodiments, an anti-FAM19A5 antibody comprises (i) a heavy chain variable domain comprising SEQ ID NO: 35 and a light chain variable domain comprising SEQ ID NO: 39; (ii) a heavy chain variable domain comprising SEQ ID NO: 36 and a light chain variable domain comprising SEQ ID NO: 40; or (iii) a heavy chain variable domain comprising SEQ ID NO: 38 and a light chain variable domain comprising SEQ ID NO: 42. In some embodiments, an anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the variable region heavy chain contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 38, and /or wherein the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence represented by SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 42.

In some embodiments, the anti-FAM19A5 antibody comprises (i) a heavy chain comprising SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 60; and (ii) a light chain comprising SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 64.

In some embodiments, the anti-FAM19A5 antibody of the present invention is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the anti-FAM19A5 antibody has one or more of the following properties:

(a) reduces, eliminates, delays and/or prevents fibrosis;

(b) reduces the formation of excess extracellular matrix (ECM);

(c) delays tumor growth or progression;

(d) binds to soluble human FAM19A5 with a KD of 10 nM or less, as measured by enzyme-linked immunosorbent assay (ELISA);

(e) binds to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA;

- 2 043381 (f) reduces, eliminates, delays and/or prevents the development of reactive gliosis;

(g) suppresses excessive proliferation of reactive astrocytes;

(h) reduces the expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2);

(j) increases the expression of c-fos and pERK in the nucleus of neurons;

(k) promotes neuronal survival;

(l) increases GAP43 expression in neurons and (m) promotes axon outgrowth.

The present invention further provides an epitope of sequence similarity member A5 19 (FAM19A5) consisting essentially of or consisting of an amino acid sequence of at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 5, 6, 9, or 10, wherein the epitope is capable of being specifically associated with a reference antibody containing (i ) a heavy chain variable domain comprising SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 38, and (ii) a light chain variable domain comprising SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO : 42.

Also provided herein is a nucleic acid encoding an antibody to FAM19A5 or an epitope of FAM19A5 described herein. Also provided is a composition comprising an anti-FAM19A5 antibody or a FAM19A5 epitope of the present invention.

In some aspects, the present invention also provides an anti-FAM19A5 antibody for treating a brain tumor.

In other aspects, the present invention provides a method of diagnosing a subject in need thereof, comprising contacting a biological sample of the subject with an anti-FAM19A5 antibody described herein.

Embodiments of the invention

Embodiment 1. An isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human sequence similarity 19 family member A5 (FAM19A5) and exhibits one or more of the following properties:

(a) reduces, eliminates, delays and/or prevents fibrosis;

(b) reduces the formation of excess extracellular matrix (ECM);

(c) delays tumor growth or progression.

Embodiment 2. The monoclonal antibody or antigen-binding portion thereof of Embodiment 1, which further comprises one or more of the following properties:

(d) binds to soluble human FAM19A5 with a K _D of 10 nM or less, as measured by enzyme-linked immunosorbent assay (ELISA);

(e) binds to membrane-bound human FAM19A5 with a K _D of 10 nM or less, as measured by ELISA;

(f) reduces, eliminates, delays and/or prevents the development of reactive gliosis;

(g) suppresses excessive proliferation of reactive astrocytes;

(h) reduces the expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2);

(j) increases the expression of c-fos and pERK in the nucleus of neurons;

(k) promotes neuronal survival;

(l) increases GAP43 expression in neurons;

(m) promotes axon regrowth.

Embodiment 3. The monoclonal antibody or antigen binding portion thereof of Embodiment 1 or 2, which competes for binding to a human FAM19A5 epitope with (i) a reference antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein Heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 11, heavy chain CDR2 contains the amino acid sequence of SEQ ID

NO: 12, CDR3 heavy chain amino acid amino acid sequence sequence sequence

SEQ ID

SEQ ID

NO:

NO:

SEQ ID NO:

13,

23, and

CDR1

CDR2

CDR3 light light light chain chain contains contains contains contains amino acid sequence SEQ ID NO: 25; (ii) with a reference antibody containing CDR1,

CDR2 and CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain, where CDR1 of the heavy chain contains

amino acid subsequence SEQ ID NO: 14, CDR2 severe chains contains amino acid subsequence SEQ ID NO: 15, CDR3 severe chains contains amino acid subsequence SEQ ID NO: 16, CDR1 easy chains contains amino acid subsequence SEQ ID NO: 26, CDR2 easy chains contains amino acid subsequence SEQ ID NO: 27 and CDR3 easy chains contains

sequence SEQ ID NO: 28; or (iii) with an amino acid reference antibody containing

CDR1, CDR2 and CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain, where CDR1 of the heavy chain contains - 3 043381

live amino acid sequence SEQ ID NO: 20, CDR2 severe chains contains amino acid subsequence SEQ ID NO: 21, CDR3 severe chains contains amino acid subsequence SEQ ID NO: 22, CDR1 easy chains contains amino acid subsequence SEQ ID NO: 32, CDR2 easy chains contains amino acid subsequence SEQ ID NO: 33 and CDR3 easy chains contains

amino acid sequence SEQ ID NO: 34.

Embodiment 4. The monoclonal antibody or antigen binding portion thereof of Embodiment 1 or 2 that binds to the same FAM19A5 epitope as (i) the reference antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 wherein the heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 11, the heavy chain CDR2 co-

holds the amino acid sequence SEQ ID NO: 12, CDR3 severe chains contains amino acid subsequence SEQ ID NO: 13, CDR1 easy chains contains amino acid subsequence SEQ ID NO: 23, CDR2 easy chains contains amino acid subsequence SEQ ID NO: 24 and CDR3 easy chains contains amino acid sequence SEQ ID NO: 25; (ii) as a reference antibody containing CDR1, CDR2 and CDR3 heavy chain and CDR1, CDR2 and CDR3 of the light chain, where CDR1 of the heavy chain contains amino acid subsequence SEQ ID NO: 14, CDR2 severe chains contains amino acid subsequence SEQ ID NO: 15, CDR3 severe chains contains amino acid subsequence SEQ ID NO: 16, CDR1 easy chains contains amino acid subsequence SEQ ID NO: 26, CDR2 easy chains contains amino acid subsequence SEQ ID NO: 27 and CDR3 easy chains contains amino acid SEQ ID sequence NO: 28; or (iii) as a reference antibody containing

CDR1, CDR2 and CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain, where CDR1 of the heavy chain

contains the amino acid sequence SEQ ID NO: 20, CDR2 of the heavy chain contains amino acid subsequence SEQ ID NO: 21, CDR3 severe chains contains amino acid subsequence SEQ ID NO: 22, CDR1 easy chains contains amino acid subsequence SEQ ID NO: 32, CDR2 easy chains contains amino acid subsequence SEQ ID NO: 33 and CDR3 easy chains contains

amino acid sequence SEQ ID NO: 34.

Embodiment 5. The monoclonal antibody or antigen binding portion thereof of Embodiment 3 or 4 that binds to at least one epitope of FAM19A5 that is SEQ ID NO: 6.

Embodiment 6. The monoclonal antibody or antigen-binding portion thereof of any one of embodiments 2-5, wherein the monoclonal antibody or antigen-binding portion thereof binds only to FAM19A5, which is SEQ ID NO: 6.

Embodiment 7. The monoclonal antibody or antigen-binding portion thereof of any one of embodiments 2-5, wherein the monoclonal antibody or antigen-binding portion thereof further binds to an additional epitope of FAM19A5.

Embodiment 8. The monoclonal antibody or antigen binding portion thereof of Embodiment 7, wherein the additional FAM19A5 epitope is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and any combination thereof.

Embodiment 9. The monoclonal antibody or antigen binding portion thereof of any one of embodiments 1 to 8, which comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3.

Embodiment 10. The monoclonal antibody or antigen binding portion thereof of Embodiment 9, wherein the heavy chain CDR1 comprises SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 20.

Embodiment 11. The monoclonal antibody or antigen binding portion thereof of Embodiment 9 or 10, wherein the heavy chain CDR2 comprises SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 21.

Embodiment 12. The monoclonal antibody or antigen binding portion thereof of any one of embodiments 9-11, wherein the heavy chain CDR3 comprises SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 22.

Embodiment 13. The monoclonal antibody or antigen binding portion thereof of any one of embodiments 9-12, wherein the light chain CDR1 comprises SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 32.

Embodiment 14. The monoclonal antibody or antigen binding portion thereof of any one of embodiments 9-13, wherein the light chain CDR2 comprises SEQ ID NO: 24, SEQ ID NO: 27, or SEQ ID NO: 33.

Embodiment 15. The monoclonal antibody or antigen binding portion thereof of any one of embodiments 9-14, wherein the light chain CDR3 comprises SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO: 34.

Embodiment 16. A monoclonal antibody or an antigen-binding portion thereof according to any one

- 4 043381 of embodiments 1-15, which contains (i) a heavy chain variable domain containing

SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 38, and (ii) a light chain variable domain containing

SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 42.

Embodiment 17. The monoclonal antibody or antigen-binding portion thereof of any one of embodiments 1-16, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least at least about 99% or about 100% identical to the amino acid sequence shown as SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 38.

Embodiment 18. The monoclonal antibody or antigen-binding portion thereof of any one of embodiments 1-17, which comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least about 80% , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, according to at least about 99% or about 100% identical to the amino acid sequence represented by SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 42.

Embodiment 19. The monoclonal antibody of any one of embodiments 1-18, wherein the monoclonal antibody is a single domain antibody.

Embodiment 20. The monoclonal antibody or antigen-binding portion thereof of any one of embodiments 1 to 19, wherein the monoclonal antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, a variant thereof, or a combination thereof.

Embodiment 21. The monoclonal antibody or antigen-binding portion thereof of Embodiment 20, wherein the monoclonal antibody is a human IgG1 antibody.

Embodiment 22. The monoclonal antibody or antigen-binding portion thereof of Embodiment 20, wherein the monoclonal antibody comprises an IgG2 or IgG4 human antibody isotype.

Embodiment 23. The monoclonal antibody or antigen-binding portion thereof of any one of embodiments 1-22, further comprising a constant region without Fc function.

Embodiment 24. The monoclonal antibody or antigen-binding portion thereof of any one of embodiments 1-23, which is a chimeric antibody, a human antibody, or a humanized antibody.

Embodiment 25. The monoclonal antibody of any one of embodiments 1-24, wherein the monoclonal antibody comprises (i) a heavy chain comprising SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 60; and (ii) a light chain comprising SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 64.

Embodiment 26. The monoclonal antibody or antigen binding portion thereof of any one of embodiments 1 to 25, wherein the antigen binding portion thereof is a Fab, Fab', F(ab') ₂ , Fv, or single chain Fv (scFv).

Embodiment 27. A bispecific molecule comprising a monoclonal antibody or an antigen binding moiety thereof according to any one of embodiments 1-26, which is linked to a molecule having a second binding moiety.

Embodiment 28. An epitope of human sequence similarity 19 family member A5 (FAM19A5), consisting essentially of, or consisting of, an amino acid sequence of at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 5, 6, 9, or 10, wherein the epitope is capable of being specifically linked to the reference antibody comprising (i) a heavy chain variable domain comprising SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 38, and (ii) a light chain variable domain comprising SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 42.

Embodiment 29. A nucleic acid encoding a monoclonal antibody or an antigen-binding portion thereof as in any of Embodiments 1-26, a bispecific molecule as in Embodiment 27, or an epitope as in Embodiment 28.

Embodiment 30. A vector containing the nucleic acid of embodiment 29.

Embodiment 31. The vector of embodiment 30 for use in gene therapy.

Embodiment 32. A cell transformed with the expression vector of Embodiment 30.

Embodiment 33. An immunoconjugate comprising a monoclonal antibody or an antigen-binding portion thereof according to any one of embodiments 1-26, or a bispecific molecule according to

- 5 043381 embodiment 27, associated with the agent.

Embodiment 34. A composition comprising (i) a monoclonal antibody or antigen-binding portion thereof as in any one of embodiments 1-26, a bispecific molecule as in embodiment 27, or an immunoconjugate as in embodiment 33, and (ii) a carrier.

Embodiment 35. A kit comprising (i) a monoclonal antibody or antigen-binding portion thereof as in any one of embodiments 1-26, a bispecific molecule as in embodiment 27, or an immunoconjugate as in embodiment 33, and (ii) instructions for use.

Embodiment 36. A method of preparing an antibody to FAM19A5 or an antigen-binding portion thereof, comprising immunizing a non-human animal with the epitope of Embodiment 28 and producing the antibody or antigen-binding portion thereof.

Embodiment 37. A method for producing an antibody to FAM19A5 or an antigen-binding portion thereof, comprising culturing the cell of Embodiment 32 under suitable conditions and isolating the antibody or antigen-binding portion thereof.

Embodiment 38. A method for reducing, eliminating, and/or preventing fibrosis in a subject in need thereof, comprising administering a monoclonal antibody or antigen-binding portion thereof of any one of embodiments 1-26, a bispecific molecule of embodiment 27, or an immunoconjugate of embodiment 33, in such a way that the development of fibrosis is delayed.

Embodiment 39. A method of reducing the formation of excess extracellular matrix (ECM) in a subject in need thereof, comprising administering to the subject a monoclonal antibody or an antigen-binding portion thereof of any one of Embodiments 1-26, a bispecific molecule of Embodiment 27, or an immunoconjugate of Embodiment 33 , where the formation of excess ECM is reduced.

Embodiment 40. A method for preventing, ameliorating, or treating liver cancer, lung cancer, kidney cancer, breast cancer, and/or pancreatic cancer in a subject in need thereof, comprising administering to the subject a monoclonal antibody or an antigen-binding moiety thereof according to any one of embodiments. 1-26, the bispecific molecule of embodiment 27, or the immunoconjugate of embodiment 33.

Embodiment 41. The method of Embodiment 40, wherein the liver cancer is hepatocellular carcinoma.

Embodiment 42. A method for preventing, ameliorating, or treating liver cancer, lung cancer, kidney cancer, breast cancer, and/or pancreatic cancer associated with fibrosis in a subject in need thereof, comprising administering to the subject a monoclonal antibody or an antigen-binding portion thereof at any of embodiments 1-26, the bispecific molecule of embodiment 27, or the immunoconjugate of embodiment 33.

Embodiment 43. The method of Embodiment 38 or 42, wherein the fibrosis includes liver fibrosis, pulmonary fibrosis, renal fibrosis, myelofibrosis, pancreatic fibrosis, skin fibrosis, myocardial fibrosis, arterial fibrosis, arthrofibrosis, mammary fibrosis, muscular fibrosis, retroperitoneal fibrosis , thyroid fibrosis, lymph node fibrosis, bladder fibrosis and/or pleural fibrosis.

Embodiment 44. The method of any one of embodiments 38-43, wherein the monoclonal antibody or antigen-binding portion thereof is administered intravenously, orally, parenterally, intrathecally, intracerebroventricularly, pulmonaryly, subcutaneously, or intraventricularly.

Embodiment 45. The method as in any one of embodiments 38-44, wherein the subject is a human.

Embodiment 46. A method for measuring the level of FAM19A5 in a sample from a subject in need thereof, comprising contacting the monoclonal antibody or antigen binding portion thereof of any one of Embodiments 1-26, the bispecific molecule of Embodiment 27, or the immunoconjugate of Embodiment 33 with the sample obtained from the subject.

Embodiment 47. The method of Embodiment 46, wherein the level of FAM19A5 is increased in the subject sample compared to the control sample.

Embodiment 48. A method for diagnosing fibrosis in a subject in need thereof, comprising contacting the monoclonal antibody or antigen-binding portion thereof of any one of embodiments 1-26, the bispecific molecule of embodiment 27, or the immunoconjugate of embodiment 33 with a sample obtained from the subject.

Embodiment 49. The method of Embodiment 48, wherein the fibrosis is fibrosis of the lung or liver.

Embodiment 50. The method of Embodiment 48 or 49, wherein the level of FAM19A5 is increased in the subject sample compared to the level of FAM19A5 in the control sample from the subject without fibrosis.

Embodiment 51. A method for diagnosing a fibrotic tumor in a subject in need thereof, comprising contacting a monoclonal antibody or antigen-binding portion thereof at any one of

- 6 043381 embodiments 1 to 26, the bispecific molecule of embodiment 27, or the immunoconjugate of embodiment 33 with a sample obtained from a subject.

Embodiment 52. The method of Embodiment 51, wherein the level of FAM19A5 is increased in the subject sample compared to the level of FAM19A5 in the control sample of the subject without a tumor.

Embodiment 53. The method as in any one of embodiments 46-52, wherein the sample is blood, serum, plasma, cerebrospinal fluid, tissue biopsy, organ biopsy, or a combination thereof.

Embodiment 54. The method of any one of embodiments 46-52, wherein the sample is a fresh tumor sample, a frozen tumor sample, or a formalin-fixed, paraffin-embedded sample.

Embodiment 55. The method as in any one of embodiments 46-52, wherein the contact includes performing an analysis.

Embodiment 56. The method of Embodiment 54, wherein the analysis includes Western blot analysis, slot blot analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry-based immunofluorescence analysis (FACS), mass spectrometry, surface plasmon resonance (SPR) or a combination of both.

Brief description of graphic materials

Fig. 1 shows the amino acid sequences of the F1-F6 epitopes (conjugated to BSA) and their location on the human FAM19A5 polypeptide. The sizes of the various epitope fragments are indicated in parentheses.

Fig. 2 shows ELISA results for binding antibodies to FAM19A5 1-65, 2-13 and 3-2. Antibody 3-2 showed two different isotypes: human IgG1 (h3-2) and mouse IgG1 (m3-2). For each antibody, the 1st, 2nd, 3rd, 4th, 5th and 6th columns (starting from the left) represent binding to epitope fragments F1, F2, F3, F4, F5 and F6, respectively.

The rightmost column displays the positive control (i.e., His-tagged FAM19A5 protein). Exact O.D. values are indicated at the top of each column.

Fig. 3A-3C show ELISA results for the binding of anti-FAM19A5 antibodies 3-2 and 1-28 to thirteen different mutant peptides of the F2 epitope fragment of FAM19A5: (i) F2-01-BSA (#1), (ii) F2-02-BSA ( #2), (iii) F2-03-BSA (#3), (iv) F2-04-BSA (#4), (v) F2-05-BSA (#5), (vi) F2-06- BSA (#6), (vii) F2-07-BSA (#7), (viii) F2-08-BSA (#8), (ix) F2-09-BSA (#9), (x) F2- 10-BSA (#10), (xi) F2-11-BSA (#11), (xii) F2-12-BSA (#12), and (xiii) F2-13-BSA (#13).

Fig. 3A shows results for antibody 3-2 with the human IgG1 isotype.

Fig. 3B shows results for antibody 3-2 with the murine IgG1 isotype.

Fig. 3C shows results for antibody 1-28.

The exact OD values are indicated at the top of each column.

Fig. 4 shows the amino acid sequence alignment of different members of the FAM19A family (ie, FAM19A1-5). Regions with the greatest amino acid diversity among members are indicated by boxes and shown as EP1-EP8. IgG antibody (IgG only) is shown as a control. Y axis provides OD value

Fig. 5 shows ELISA results for binding of anti-FAM19A5 antibodies 1-65, 1-28, 2-13 and 3-2 to FAM19A5 mutants M1-M8. For each antibody, eight columns correspond to mutants M1-M8 (moving from left to right).

Fig. 6A shows a schematic diagram of a two-site sandwich ELISA assay that is used to evaluate competition among different antibodies to FAM19A5.

Fig. 6B shows the results of a competition assay for six different antibodies to FAM19A5: 1-65, P2-A03, P2-F11, 13B4, 2-13 and 3-2. The term S/N refers to the signal to noise ratio, which is measured as follows: [OD 10 ng/ml antigen]/[OD 0 ng/ml antigen]. Gray rectangles indicate competitiveness (i.e. S/N ratio lower than 2).

Fig. 7A and 7B show ELISA results for binding of several anti-FAM19A5 antibodies to FAM19A5. Results are shown for the following antibodies: 1-65, 13B4, 13F7, 1-28,2-13, 3-2, P1-A03, P1-A08, P1-D03, P1-F02, P1-G09, P2-A01, P2-A03, P2-C12, P2-F07 and P2-F11 (moving from left to right).

Fig. 7A shows the results as a bar graph for different concentrations of anti-FAM19A5 antibodies.

Fig. 7B shows Kd (nM) for different antibodies to FAM19A5.

Fig. 8 shows a comparison of FAM19A5 protein levels in the serum of rats (n=5) with bile duct ligation (BDL)-induced liver fibrosis (disease model) versus healthy rats (normal) (n=3). FAM19A5 protein levels are shown as fold change compared to the levels observed in normal control animals. *Denotes statistically significant difference (p<0.005) compared to normal controls.

Fig. 9 shows a comparison of FAM19A5 protein levels in the serum of rats (n=5) with idiopathic pulmonary fibrosis induced by intrathecal injection of 3 mg/kg bleomycin (disease model) versus healthy rats (normal) (n=5). FAM19A5 protein levels are shown as fold change

- 7 043381 compared to the level observed in normal control animals. *Denotes statistically significant difference (p<0.005) compared to normal controls.

Fig. 10 shows the fold change in FAM19A5 protein levels in the serum of both patients with confirmed liver fibrosis, such as cirrhosis, (patient #1-10) and healthy individuals (normal #1-3). The specific value indicated above each histogram bar denotes the fold change in the mean concentration of FAM19A5 protein found in the serum of healthy individuals.

Fig. 11 shows a comparison of H&E (hematoxylin and eosin) stained left ventricular tissue from animals with induced myocardial infarction treated with control antibody (NHI) or anti-FAM19A5 antibody (1-65, 3-2 and 1-28). Healthy animals (i.e., without induced myocardial infarction, naïve) were used as additional controls.

Fig. 12A and 12B show a comparison of collagen accumulation in left ventricular tissue in animals with induced myocardial infarction. Animals with induced myocardial infarction received either control antibody (NHI) or anti-FAM19A5 antibody (1-65, 3-2 and 1-28). Healthy animals (i.e., without induced myocardial infarction, naïve) were again used as additional controls.

Fig. 12A demonstrates collagen accumulation using Masson's trichrome stain.

Fig. 12B shows collagen accumulation (ie, area of fibrosis) as a ratio of area of fibrosis to area of the entire left ventricle. Data are expressed as arithmetic mean ± S.D (n=5-8 per group). **/** Above the bars indicates a statistically significant difference (p<0.001, p<0.05, respectively) compared to the naive group (healthy animals). The # above the bars denotes a statistically significant difference (p<0.05) compared to the NHI group (induced myocardial infarction + control antibody).

Fig. 13 shows immunohistochemical analysis of FAM19A5 protein expression in liver biopsies from three different liver cancer patients. As indicated, each of the patients had varying degrees of fibrosis: (i) stage #0 (left column), (ii) stage #2 (middle column), and (iii) stage #4 (right column). The bottom row shows a greater magnification of the boxed area from the top row. Arrows indicate examples of FAM19A5-positive hepatic stellate cells.

Fig. 14A shows the body weight (grams) of animals as a function of time (weeks after inoculation). Some animals were inoculated with Hep3B cells only and treated with normal human immunoglobulin (circle, group: Hep3B + NHI, n=3) or anti-FAM19A5 antibody (solid box, group: Hep3B + FAM19A5 Ab, n=3). Other animals were inoculated with both Hep3B cells and human liver stellate cells (HHSteC) and treated with normal human immunoglobulin (diamond, group: Hep3B + HHSteC + NHI, n=3) or anti-FAM19A5 antibody (unfilled rectangle, group: Hep3B + HHSteC + FAM19A5 Ab, n=3). Data are expressed as arithmetic mean ± S.D.

Fig. 14B shows the average tumor volume observed in animals from different groups as a function of time (weeks after inoculation). These groups are the same as those described in FIG. 14A. Tumor volume was calculated according to the following formula: 0.5 x length x width ² = tumor volume (mm ³ ). Data are expressed as arithmetic mean ± SD

Fig. 14C and 14D show photographic images and weights (grams), respectively, of tumors isolated from animals as described in FIG. 14A, on day 42 after inoculation.

In fig. 14C, (i) top left shows tumors from the Hep3B + NHI group (n=2); (ii) top right shows tumors from the Hep3B + HHSteC + NHI group (n=2); (iii) bottom left shows tumors from the Hep3B + FAM19A5 Ab group (n=3) and (iv) bottom right shows tumors from the Hep3B + HHSteC + FAM19A5 Ab group (n=3).

Fig. 14D shows the average weight (g) of tumors isolated from different groups.

Fig. 15A and 15B demonstrate the effect of antibodies to FAM19A5 on reactive gliosis after traumatic brain injury.

Fig. 15A provides representative immunohistochemical images of a damaged area of brain tissue from animals treated with various anti-FAM19A5 antibodies: (i) 1-65 (2/4) (1st row); (ii) 1-28 (2nd row); (iii) 2-13 (3rd row) and (iv) 3-2 (4th row). Brain tissue sections were stained for GFAP (glial fibrillary acidic protein, green) and nestin (red), which are known to be induced in reactive astrocytes after brain injury. The dotted line (in white) denotes the damage boundary after TBI exposure.

Fig. 15B provides the average distance of GFAP- and/or nestin-positive astrocytes from the center of the TBI lesion in animals treated with 1-65, 1-28, 2-13, and 3-2 anti-FAM19A5 antibodies.

Fig. 16A provides both a schematic of antibody administration and a circuit diagram demonstrating intracranial injection of glioblastoma cancer cells into mice.

Fig. 16B demonstrates the antitumor effect of anti-FAM19A5 antibody in animal models of glioblastoma using CLARITY, which assesses the transparency of a tissue sample. Regions

- 8 043381 tumors that appear opaque are indicated by dotted lines in animals from which brain tissue treated with either control human IgG antibody (left) or anti-FAM19A5 antibody (right) was collected.

Fig. 17A and 17B demonstrate the antitumor effect of anti-FAM19A5 antibody in animal models of glioblastoma using hematoxylin and eosin (H&E) staining.

Fig. 17A provides four representative images of H&E staining of animal brain tissue sections treated with either control human IgG antibody (top row) or anti-FAM19A5 antibody (bottom row). Glioblastoma corresponds to the darkened areas on the images.

Fig. 17B represents the data shown in FIG. 17A, in quantitative form, providing data in percentage control. Black bars correspond to the control group (treated with human IgG antibody). Gray bars correspond to the anti-FAM19A5 antibody-treated group. The numbers along the x-axis correspond to the representative image shown in FIG. 17A.

Fig. 18A-18D demonstrate the antitumor effect of anti-FAM19A5 antibody in an animal model of glioblastoma using Hoechst nuclear staining.

Fig. 18A shows an image of four representative sections of animal brain tissue treated with control human IgG antibody (top row) or anti-FAM19A5 antibody (bottom row). The areas shown in light gray correspond to the tumor in each image.

Fig. 18B is a table showing numerical values for tumor cell number (Hoechst positive staining) (Spot No.) and tumor volume (Vol.) measured in four representative sections of brain tissue in FIG. 18A.

Fig. 18C (cell number) and 18D (tumor volume) are graphical representations of the data shown in FIG. 18V. As in fig. 18C, and in FIG. 18D, data are presented as % of the corresponding value observed in control animals. The numbers along the x-axis correspond to a representative section of brain tissue from Fig. 18A. Black bars correspond to animals treated with control human IgG antibody. Gray bars correspond to animals treated with anti-FAM19A5 antibody.

Fig. 19A and 19B demonstrate the effect of anti-FAM19A5 antibody on normalizing blood vessels in a mouse model of glioblastoma.

Fig. 19A shows immunohistochemical analysis of CD31 expression (a blood vessel marker) in matched brain regions isolated from animals treated with control human IgG antibody (left image) or anti-FAM19A5 antibody (right image). The dotted white lines in Fig. 19A represents the outer border of a glioblastoma.

Fig. 19B is an enlarged view of a representative area of FIG. 19A.

Fig. 20A and 20B demonstrate the effect of anti-FAM19A5 antibody on macrophage infiltration into tumors in a mouse model of glioblastoma.

Fig. 20A shows an immunohistochemical image of Iba1 expression (a marker for macrophages) in glioblastoma from mice treated with control IgG antibody (upper image) or anti-FAM19A5 antibody (lower image).

Fig. 20B compares the volume of macrophages observed in the images shown in FIG. 20A.

Fig. 21 shows MPT scans of brains from rat glioma models treated with either control human IgG antibody (top row) or anti-FAM19A5 antibody (bottom row). Images demonstrate various properties of tumors: (i) T2 - morphological analysis; (ii) R2 - morphological analysis; (iii) CBV - cerebral blood volume; (iv) EnhRate (expansion rate) and IAUC30 (initial area under the curve at 30 s) - tissue permeability. The tumor is indicated on the images by black dotted marks.

Fig. 22 demonstrates the effect of anti-FAM19A5 antibody on survival in a mouse model of brain tumor. Black circles represent animals treated with control human IgG antibody. White circles represent animals treated with anti-FAM19A5 antibody.

Detailed Description of the Invention

An isolated monoclonal antibody, or an antigen-binding portion thereof, described herein that specifically binds to human sequence similarity 19 family member A5 (FAM19A5) and exhibits one or more of the properties described herein, such as reducing, eliminating, delaying, and/or preventing fibrosis; reduces the formation of excess extracellular matrix (ECM); delays tumor growth or progression; binds to soluble human FAM19A5 with a KD of 10 nM or less, as measured by enzyme-linked immunosorbent assay (ELISA); binds to membrane-bound human FAM19A5 with a KD of 10 nM or less as measured by ELISA; reduces, eliminates, delays and/or prevents the development of reactive gliosis; suppresses excessive proliferation of reactive astrocytes; reduces the expression of chondroitin sulfate proteoglycans, including neurocan and neuron-glial antigen 2 (NG2); increases the expression of c-fos and pERK in the nucleus of neurons; promotes neuronal survival; increases the expression of GAP43 in neurons; and/or promotes axon regrowth.

To facilitate understanding of the present invention, a number of terms and phrases are defined. Additional definitions are provided in the detailed description.

I. Definitions.

Throughout this specification, the singular form of object refers to one or more such objects; for example, an antibody is understood as one or more antibodies. As such, the terms singular, one or more, and at least one may be used interchangeably herein. Furthermore, and/or as used herein, should be understood as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term and/or used in a phrase such as A and/or B as used herein is intended to include A and B, A or B, A (alone) and B (alone). It is also intended that the term and/or used in a phrase such as A, B and/or C cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separately) and C (separately). It is understood that wherever aspects are described herein in terms of containing, in other cases similar aspects are also described in terms of consisting of and/or consisting substantially of.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as would be known to one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, ^2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, ^3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one skilled in the art with a general vocabulary of many of the terms used herein.

Units, prefixes, and symbols are designated in the accepted form of the International System of Units (SI). Numeric ranges include numbers that define a range. Unless otherwise noted, amino acid sequences are written from left to right in amino-carboxy orientation. The headings provided herein are not intended to limit the various aspects of the description that may be derived by reference to the description as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

The term approximately is used herein to mean about, about, approximately, or in the area. When a term is approximately used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. Generally, a term approximately may vary in numerical value above and below the stated value with a deviation of, for example, 10%, up or down (above or below).

The term family sequence similarity member A5 19 or FAM19A5 refers to a protein that belongs to the TAFA family (also known as the FAM19 family) of five highly homologous proteins and is predominantly expressed in the brain and spinal cord. FAM19A5 is also known as TAFA5 or chemokine-like protein TAFA-5. In humans, the gene encoding FAM19A5 is located on chromosome 22.

There are three human isoforms of FAM19A5 (UniProt: Q7Z5A7), which are thought to be generated by alternative splicing: isoform 1 (UniProt: Q7Z5A7-1), which consists of 132 amino acids; isoform 2 (UniProt: Q7Z5A7-2), which consists of 125 amino acids; and isoform 3 (UniProt: Q7Z5A7-3), which consists of 53 amino acids. The human FAM19A5 protein is believed to exist in both membrane-bound and soluble (secreted) forms. Isoform 1 is predicted to be a membrane-bound protein with a single transmembrane region. Isoform 2, reported in Tang T.Y. et al., Genomics, 83(4):727-34 (2004) as a secreted protein (soluble), contains a signal peptide at amino acid positions 1-25. Isoform 3 predicted from EST data. Below are the amino acid sequences of the three known human isoforms of FAM19A5.

(I) Isoform 1 (UniProt: Q7Z5A7-1, transmembrane protein): this isoform was chosen as the canonical sequence:

MAPSPRTGSR QDATALPSMS STFWAFMILA SLLIAYCSQL AAGTCEIVTL

DRDSSQPRRT IARQTARCAC RKGQIAGTTR ARPACVDARI IKTKQWCDML PCLEGEGCDL LINRSGWTCT QPGGRIKTTT VS (SEQ ID NO: 1) (II) Isoform 2 (UniProt: Q7Z5A7-2, soluble protein):

MQLLKALWAL AGAALCCFLV LVIHAQFLKE GQLAAGTCEI

VTLDRDSSQP RRTIARQTAR CACRKGQIAG TTRARPACVD ARIIKTKQWC

DMLPCLEGEG CDLLINRSGW TCTQPGGRIK TTTVS (SEQ ID NO: 2)

- 10 043381 (III) Isoform 3 (UniProt: Q7Z5A7-3):

MYHHREWPAR IIKTKQWCDM LPCLEGEGCD LLINRSGWTC

TQPGGRIKTT TVS (SEQ ID NO: 3)

The term FAM19A5 includes any variants or isoforms of FAM19A5 that are naturally expressed by cells. Accordingly, the antibodies described herein may cross-react with different isoforms of the same species (eg, different isoforms of human FAM19A5) or cross-react with FAM19A5 of non-human species (eg, murine FAM19A5). Alternatively, the antibodies may be specific for human FAM19A5 and may not be cross-reactive with other species. FAM19A5 or any variants and isoforms thereof can be isolated from cells or tissues that naturally express them or can be produced recombinantly. The polynucleotide encoding human FAM19A5 has GenBank accession number BC039396 and the following sequence (Table 1A).

Table 1A

Polynucleotide sequence of human FAM19A5

Polynucleotide sequence (SEQ ID NO: 4) FAM19A5 (GenBank accession number ВС039396) ggcggcggag gatggcgcgc gcggggcccg cacgtggagg ccggcgcggg ggcgcgggca gggccggctg ctgagacgcg ctgctgcccc ccgcgcgggc gccgcggctt caatggcgcc atcgcccagg accggcagcc ggcaagatgc gaccgccctg cccagcatgt cctca acttt ctgggcgttc atgatcctgg ccagcctgct catcgcctac tgcagtcagc tggccgccgg cacctgtgag attgtgacct tggaccgga cagcagccag cctcggagga cgatcgcccg gcagaccgcc cgctgtgcgt gtagaaaggg gcagatcgcc ggcaccacga gagcccgg cc cgcctgtgtg gacgcaagaa tcatcaagac caagcagtgg tgtgacatgc ttccgtgtct ggagggggaa ggctgcgact tgttaatcaa ccggtcaggc tggacgtgca cgcagcccgg cgggaggata aagaccacca cggtctcctg acaaacacag cccctgaggg ggccccggga gtggccttgg ctccctggag agcccacgtc tcagccacag ttctccactc gcctcggact tcacccgttc tctgccgccc gcccactccg tttccctgtg gtccgtgaag gacggcctca ggccttggca tcctgagctt cggtctgtcc agccgacccg aggaggccgg actcagacac ataggcgggg ggc ggcacct ggcatcagca atacgcagtc tgtgggagcc cggccgcgcc cagcccccgc cgaccgtggc gttggccctg ctgtcctcag aggaggagga ggaggaggca gctccggcag ccacagaagg ctgcagccca gcccgcctga gacacgacgc ctgccccagg ggactgtcag gcacagaag c ggcctcctcc cgtgccccag actgtccgaa ttgcttttat tttcttatac tttcagtata ctccatagac caaagagcaa aatctatctg aacctggacg caccctcact gtcagggtcc ctggggtcgc ttgtgcgggc ggggagggcaa tggtggcaga gacatgctgg tggccccggc ggagcggaga gggcggccgt ggtggaggcc tccaccccag gagcaccccg cacaccctcg gaggacgggc ttcggctgcg cggaggccgt ggcacacctg cgggaggcag cgacggcccc cacgcagacg ccggga acgc aggccgcttt attcctctgt acttagatca acttgaccgt actaaaatcc ctttctgttt taaccagtta aacatgcctc ttctacagct ccatttttga tagttggata atccagtatc tgccaagagc atgttgggtc tcccgtgact gctgcctcat cgatacccca tttagctcca gaaagcaaag aaaactcgag taacacttgt ttgaaagaga tcattaaatg tattttgcaa agcccaaaaa aaaaaaaaaa a

The terms antibody and antibodies are terms of the art and may be used interchangeably herein and refer to a molecule with an antigen-binding site that specifically binds an antigen. As used herein, terms include whole antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or

- 11 043381 separate chains. An antibody, in one embodiment, refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. In another embodiment, an antibody refers to a single antibody chain containing a single variable domain, such as a VH domain. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some naturally occurring antibodies, the heavy chain constant region contains three domains, CHI, CH2 and CH3. In some naturally occurring antibodies, each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain, CL.

The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The term Kabat numbering and similar terms are known in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or antigen-binding portion thereof. In certain aspects, antibody CDRs may be defined in accordance with the Kabat numbering system (see, for example, Kabat E. A. & Wutt (1971), Ann NY Acad. Sci. 190:382-391 and Kabat E. A. et al. ( 1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs in an antibody heavy chain molecule are typically present at amino acid positions 31-35, which may optionally include one or two additional amino acids following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50-65 (CDR2) and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, CDRs in an antibody light chain molecule are typically present at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been defined according to the Kabat numbering scheme.

The phrases Kabat amino acid position numbering, Kabat position, and their grammatical variants refer to the numbering system used for heavy chain variable domains or light chain variable domains of antibody compilations in Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed. . Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to the shortening or insertion of the FW or CDR variable domain. For example, a heavy chain variable domain may include one amino acid insertion (Kabat residue 52a) after residue 52 of H2 and inserted residues (eg, Kabat residues 82a, 82b and 82c, etc.) after residue 82 of the FW heavy chain. See table. 1B.

Table 1B

A loop Kabat ABM Cothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 Н31-Н35В Н26-Н35В N26-N32..34 (Numbering according to Kabat) H1 H31-H35 H26-H35 H26-H32 (Number at iya by Cothia) H2 H50-H65 H50-H58 H52-H65 NZ N95-N102 N95-N102 N95-N102

Kabat numbering of residues can be determined for a given antibody by alignment in regions of homology to the antibody sequence with standard Kabat numbering. Instead, Chothia refers to the arrangement of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the loop CDR-H1 no Chothia is considered when, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places inserts at H35A and H35B; if neither 35A nor 35B are present, then the loop ends at 32, if only 35A is present, then the loop ends at 33; if

- 12 043381 both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat's CDRs and Chothia's structural loops and are used by Oxford Molecular's AbM antibody modeling software. IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, M.P. et al., Dev. Comp. Immunol. 27:55-77(2003), which is incorporated herein by reference. The IMGT numbering system was based on over 5000 sequence alignments, structural data, and hypervariable loop characterizations and allows for easy comparison of variable regions and CDR regions across species.

According to the IMGT numbering scheme, VH-CDR1 is in positions 26-35, VH-CDR2 is in positions 51-57, VH-CDR3 is in positions 93-102, VL-CDR1 is in positions 27-32, VL-CDR2 is in positions 50-52 and VL-CDR3 is in positions 89-97. For all heavy chain constant region amino acid positions discussed herein, the numbering follows the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63(1):78-85, which describes the amino acid sequence of the EU myeloma protein, which is the first human IgG1 sequenced. EU index according to Edelman et al. also reported in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, ^5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Thus, the phrases index EU as stated by Kabat or index EU by Kabat and position ... according to index EU as stated by Kabat and their grammatical variants refer to the residue numbering system based on the human IgG1 antibody EU by Edelman et al ., as outlined in Kabat 1991.

The numbering system used for the variable domains (both heavy chain and light chain) and the amino acid sequence of the light chain constant region is presented in Kabat 1991. The antibody can be any type of immunoglobulin molecule (eg, IgG, IgE, IgM, IgD, IgA, or IgY), any class (eg, IgD, IgG2, IgG3, IgG4, IgA1 or IgA2) or any subclass (eg, IgG1, IgG2, IgG3 and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice). Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by no more than a few amino acids. The antibody described herein may be from any of the commonly known isotypes, classes, subclasses or allotypes. In certain embodiments, the antibodies described herein are antibodies of the IgG1, IgG2, IgG3 or IgG4 subclass or any hybrid thereof. In certain embodiments, the antibodies are antibodies of the human IgG1, or human IgG2, or human IgG4 subclass.

The term antibody includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; completely synthetic antibodies; single chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies containing two heavy chain molecules and two light chain molecules; antibody light chain monomer; antibody heavy chain monomer; antibody light chain dimer, antibody heavy chain dimer; antibody light chain-antibody heavy chain pair; intracellular antibodies; heteroconjugate antibodies; monovalent antibodies; camel antibodies; affitela; anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies) and single domain antibodies (sdAbs), which contain binding molecules consisting of a single monomeric antibody variable domain that are fully capable of binding to an antigen (for example, the VH domain or VL domain). Harmen M.M. and Haard H.J. Appl. Microbiol. Biotechnol. 77(1):13-22 (2007)).

The term antigen-binding portion of an antibody as used herein refers to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, human FAM19A5). Such fragments are, for example, between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably between about 8 and about 300, such as about 8 and about 200 amino acids, or about 10 to approximately 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be realized by fragments of a full-length antibody. Examples of binding fragments encompassed by the term antigen binding portion of an antibody, such as the anti-FAM19A5 antibody described herein, include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) fragment F(ab') ₂ , a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one arm of the antibody, and disulfide-linked Fvs (sdFvs); (v) a dAb fragment (Ward et al. (1989), Nature, 341:544-546), which consists of a VH domain; and (vi) a dedicated complementarity determining region (CDR), or (vii) a combination of two or more dedicated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined using recombinant methods with the help of a synthetic linker that allows them to be a single protein chain in which the VL and VH regions are combined into

- 13 043381 pair to form monovalent molecules (known as single-chain Fv (scFv)); see, for example, Bird et al. (1988), Science, 242:423-426; and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, 85:5879-5883). Such single chain antibodies are also considered to be included within the term antigen binding portion of an antibody. These antibody fragments are prepared using conventional techniques known to those skilled in the art and tested for their usefulness in the same manner as intact antibodies. Antigen-binding moieties can be obtained by recombinant DNA methods or by enzymatic or chemical digestion of intact immunoglobulins.

As used herein, the terms variable region or variable domain are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, typically a portion of the light or heavy chain, typically the amino terminus 110 to 120 amino acids in the mature heavy chain and approximately 90 to 115 amino acids in the mature light chain, which vary widely in sequence among antibodies and are used for the binding and specificity of a particular antibody for its particular antigen. Variation in the sequence is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the light and heavy chain CDRs are primarily responsible for antibody-antigen interaction and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (eg, non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (eg, non-human primate) framework regions (FRs).

As used herein, the term heavy chain, when used in reference to an antibody, can refer to any single type, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the constant domain , which is inherent in antibodies of the classes IgA, IgD, IgE, IgG and IgM, respectively, including IgG subclasses, for example IgG1, IgG2, IgG3 and IgG4.

As used herein, the term light chain, when used in relation to an antibody, can refer to any other type, such as kappa (k) or lambda (λ), based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

The terms VL and VL domain are used interchangeably to refer to the variable region of the light chain of an antibody.

The terms VH and VH domain are used interchangeably to refer to the variable region of the heavy chain of an antibody.

As used herein, the terms constant region or constant domain are used interchangeably and have a general meaning in the art. A constant region is a portion of an antibody, such as the carboxyl-terminal portion of the light and/or heavy chain, that is not directly involved in the binding of the antibody to an antigen, but which may exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule usually has a more conserved amino acid sequence relative to the variable domain of the immunoglobulin.

The Fc region (fragment of the crystallizable region) or Fc or Fc domain refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (for example, effector cells) or on the first component (C1q) of the classical complement system. Thus, the Fc region contains the antibody constant region, excluding the first constant region of the immunoglobulin domain (eg, CH1 or CL). In the IgG, IgA and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; The Fc regions of IgM and IgE contain three heavy chain constant domains (CH2-4 domains) in each polypeptide chain. For IgG, the Fc region contains the immunoglobulin domains Γγ2 and Γγ3 and the hinge between Ογ1 and Cy2. Thus, the boundaries of the immunoglobulin heavy chain Fc region can vary, the Fc region of the human IgG heavy chain is usually defined as the extent from the amino acid residue at position C226 or P230 (or the amino acid between these two amino acids) to the carboxy terminus of the heavy chain, where the numbering corresponds to the EU index , as per Kabat The CH2 domain of the Fc region of human IgG extends from approximately amino acid 231 to approximately amino acid 340, while the CH3 domain is located on the C-terminal side of the Cm domain in the Fc region, i.e. it extends from approximately 341 amino acids

- 14 043381 to 447 amino acids of IgG. The Fc region used herein may be a native Fc sequence, including any allotype or Fc variant (eg, a non-naturally occurring Fc). Fc may also refer to this region in isolation or in context

An Fc-containing protein polypeptide, such as an Fc region-containing binding protein, also called an Fc fusion protein (eg, antibody or immunoadhesion).

A native sequence Fc region or native sequence Fc region includes an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. Native Fc region sequences include the native human IgG1 Fc region sequence; the native sequence of the Fc region of human IgG2; the native human IgG3 Fc region sequence and the native human IgG4 Fc region sequence, as well as naturally occurring variants thereof. The native Fc sequence includes various Fes allotypes (see, for example, Jefferis et al. (2009), mAbs 1:1; Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014)).

The Fc receptor or FcR is a receptor that binds to the Fc region of immunoglobulin. FcRs that bind to IgG antibody include the FcyR family of receptors, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII and FcyRIV in mice; FcyRIA, FcyRIIA and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor. Human IgG1 binds to the majority of human Fc receptors and induces the most potent Fc effector functions. It is considered equivalent to mouse IgG2a in terms of the types of activating Fc receptors it binds to. On the other hand, human IgG4 induces the least Fc effector functions. Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014).

The constant region can be manipulated, for example, using recombinant technology, to eliminate one or more effector functions. Effector function refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand or the biochemical event that occurs as a result thereof. Typical effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcyR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and cell surface receptor downregulation ( e.g. B cell receptor; BCR). Such effector functions typically require that the Fc region be combined with a binding domain (eg, an antibody variable domain). Accordingly, the term constant region without Fc function includes constant regions with reduced or absent one or more effector functions mediated by the Fc region.

Antibody effector functions can be reduced or avoided using different approaches. Antibody effector functions can be reduced or avoided by using antibody fragments lacking the Fc region (eg, such as Fab, F(ab') ₂ , single chain Fv (scFv), or sdAb consisting of a monomeric VH or VL domain). Alternatively, so-called aglycosylated antibodies can be produced by removing sugars that are associated with specific residues in the Fc region to reduce the effector functions of the antibody while maintaining other valuable features of the Fc region (eg, long half-life and heterodimerization). Aglycosylated antibodies can be produced, for example, by removing or altering the residue to which the sugar is attached, enzymatic removal of sugars, production of antibodies in cells cultured in the presence of a glycosylation inhibitor, or by expression of antibodies in cells incapable of glycosylation of proteins (for example, bacterial cells - owners). See, for example, US Patent Publication No. 20120100140. Another approach is to use Fc regions from the IgG subclass that have reduced effector function, eg IgG2 and IgG4 antibodies have lower levels of Fc effector function than IgG1 and IgG3. The residues most proximal to the hinge region in the CH2 domain of the Fc region are responsible for antibody effector functions as it contains a largely overlapping binding site for C1q (complement) and IgG Fc receptors (FcyR) on effector cells of the innate immune system. Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014). Accordingly, antibodies with reduced or no Fc effector functions can be produced by creating, for example, a chimeric Fc region that contains a CH2 domain from an IgG antibody of the IgG4 isotype and a CH3 domain from an IgG antibody of the IgG1 isotype, or a chimeric Fc region that contains a hinge region from IgG2 and the CH2 region from IgG4 (see, for example, Lau C. et al., J. Immunol. 191:4769-4777 (2013)), or Fc regions with mutations that result in altered Fc effector functions , for example, reduced or absent Fc functions. Such mutated Fc regions are known in the art. See, for example, US Patent Publication No. 20120100140 and US PCT Application cited therein, and An et al., mAbs 1:6, 572-579 (2009); the description of which is incorporated herein by reference in its entirety.

The hinge, hinge domain, hinge region, or hinge region of an antibody are used interchangeably and refer to the heavy chain constant region domain that connects

- 15 043381 CH1 domain with a CH2 domain and includes the upper, middle and lower parts of the hinge (Roux et al., J. Immunol. 1998, 161:4083). The hinge provides varying levels of flexibility between the binding and effector regions of an antibody, and also provides intermolecular disulfide bond sites between two heavy chain constant regions. As used here, the hinge begins at Glu216 and ends at Gly237 for all IgGA isotypes. The hinge used herein begins at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., 1998, J. Immunol. 161:4083). The wild-type IgG1, IgG2, IgG3 and IgG4 hinge sequences are known in the art. See, for example, Kabat E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014).

The term CIS domain refers to the heavy chain constant region linking the variable domain to the hinge in the heavy chain constant region. The CH1 domain used herein begins at A118 and ends at V215. The term CIS domain includes wild-type CH1 domains as well as naturally occurring variants (eg, allotypes). The CH1 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild type and allotypes) are known in the art. See, for example, Kabat EA et al. (1991), above and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH1 domains include CH1 domains with mutations that modify the biological activity of the antibody, such as the half-life, such as those described in US patent publication. No. 20120100140, and US patents and publications, and PCT publications cited therein.

The term CH2 domain refers to the heavy chain constant region linking the hinge to the CH3 domain in the heavy chain constant domain. The CH2 domain used herein begins at P238 and ends at K340. The term CH2 domain includes wild-type CH2 domains as well as naturally occurring variants (eg, allotypes) thereof. The CH2 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild type and allotypes) are known in the art. See, for example, Kabat EA et al. (1991) above and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH2 domains include CH2 domains with mutations that modify the biological activity of the antibody, such as half-life, and/or reduce Fc effector function, such as those described in US patent publication. No. 20120100140, and US patents and publications, and PCT publications cited therein.

The term CH3 domain refers to the heavy chain constant region that is C-terminal to the CH2 domain in the heavy chain constant domain. The CH3 domain used herein begins at G341 and ends at K447. The term CH3 domain includes wild-type CH3 domains as well as naturally occurring variants (eg, allotypes) thereof. The CH3 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild type and allotypes) are known in the art. See, for example, Kabat EA et al. (1991) above and Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH3 domains include CH3 domains with mutations that modify the biological activity of the antibody, such as the half-life, such as those described in US patent publication. No. 20120100140, and US patents and publications, and PCT publications cited therein.

As used herein, an isotype refers to a class of antibodies (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) that are encoded by heavy chain constant region genes.

An allotype refers to naturally occurring variants within a specific isotype group where these variants differ in several amino acids (see, for example, Jefferis et al. (2009), mAbs 1:1). The antibodies described herein can be of any allotype. Allotypes IgG1, IgG2, IgG3 and IgG4 are known in the art. See, for example, Kabat E.A. et al. (1991) above; Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014) and Lefranc M. P., mAbs 1:4, 1-7 (2009).

The phrases antigen-recognizing antibody and antigen-specific antibody are used interchangeably herein with the term antibody that specifically binds an antigen.

An isolated antibody, as used herein, is intended to mean an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to FAM19A5 contains substantially no antibodies that specifically bind antigens other than FAM19A5). An isolated antibody that specifically binds to an epitope of FAM19A5 may be cross-reactive with other FAM19A5 proteins from different species.

Binding affinity generally refers to the strength of the total number of noncovalent interactions between one binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified as used herein, binding affinity refers to the true binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of molecule X for its

- 16 043381 partner Y can usually be represented by a dissociation constant (KD). Affinity can be measured and/or expressed in several ways known in the art, including, but not limited to, the equilibrium dissociation constant (K _D ) and the equilibrium association constant (KA). K _D is calculated from the coefficient koff/ko _n and is expressed as molar concentration (M), while KA is calculated from the coefficient k _on /ko _ff . k _on and refers to the rate constant of association, e.g. antibody to antigen, ak _off refers to dissociation, e.g. antibody from antigen. ko _n and ko _ff can be determined by methods known to one skilled in the art, such as immunoassays (eg, enzyme-linked immunosorbent assay (ELISA)), BIACORE® or kinetic exclusion assay (KINEXA®).

As used herein, the terms specifically binds, specifically recognizes, specific binds, selectively binds, and selectively binds are similar terms in the context of antibodies and refer to molecules (eg, antibodies) that bind to an antigen (eg, epitope or immune complex) because such binding will be understood by one skilled in the art. For example, a molecule that specifically binds an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined, for example, by immunoassays, BIACORE®, KINEXA® 3000 instrument (Sapidyne Instruments, Boise, Idaho) or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 log, 2.5 log, 3 log, 4 log, or greater than the KA when the molecules bind to another antigen.

Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (K _D ) of 10 ^-5 to 10 ^-11 M or less. Any KD greater than 10 ^-4 M is generally considered to indicate nonspecific binding. As used herein, an antibody that specifically binds to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means that the KD is 10 ^-7 M or less, preferably 10 ^-8 M or less, even more preferably 10 ^-9 M or less and most preferably between 10 ^-8 and 10 ^-10 M or less, when determined, for example, using immunoassays (for example, ELISA) or surface plasmon resonance (SPR) technology on a BIACORE™ 2000 instrument, using a predetermined antigen, but which does not bind with high affinity to unrelated antigens.

As used herein, the term antigen refers to any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. The antigen may be FAM19A5 or a fragment thereof.

As used herein, epitope is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. The epitope may be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope) or the epitope may, for example, be combined from two or more non-contiguous regions of the polypeptide or polypeptides (conformational, non-linear, discontinuous or non-contiguous epitope). Epitopes formed from contiguous amino acids are usually, but not always, preserved when exposed to denaturing solvents, whereas epitopes formed from tertiary stacking are usually destroyed when treated with denaturing solvents. An epitope typically contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which overlapping or adjacent peptides (eg, from FAM19A5) are tested for reactivity with a given antibody (for example, anti-FAM19A5 antibody). Methods for determining the spatial conformation of epitopes include methods in the art and methods described herein, such as X-ray crystallography, 2-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). In certain embodiments, the epitope to which the antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen-deuterium exchange mass spectrometry (e.g., electrospray liquid chromatography mass spectrometry) , array-based oligopeptide scanning analysis and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any of the methods known in the art (eg, Giege R. et al. (1994), Acta Crystallogr. D Biol. Crystallogr. 50(Pt 4):339-350; McPherson A (1990), Eur J Biochem 189:123; Chayen N E (1997), Structure, 5:1269-1274; McPherson A (1976), J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known x-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.;

- 17 043381 see, for example, Meth. Enzymol. (1985), volumes 114 & 115, eds Wyckoff H.W. et al.; U.S. 2004/0014194) and BUSTER (Bricogne G. (1993), Acta Crystallogr. D Biol. Crystallogr. 49(Pt 1):37-60; Bricogne G. (1997), Meth. Enzymol. 276A:361-423, ed Carter CW; Roversi P et al (2000), Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be performed using any method known to one skilled in the art. See, for example, Champe M. et al. (1995), J. Biol. Chem. 270:1388-1394 and Cunningham V.S. & Wells J.A. (1989), Science, 244:1081-1085 for a description of mutagenesis methods, including alanine scanning mutagenesis methods.

The term epitope mapping refers to the process of identifying molecular determinants for antibody-antigen recognition.

The term binds to the same epitope with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by this method. Methods for determining whether antibodies bind to the same epitope on FAM19A5 as the antibodies described herein include, for example, epitope mapping techniques such as crystal X-ray crystal analysis of antigen:antibody complexes, which provides atomic separation of the epitope, and mass hydrogen-deuterium exchange spectrometry (HDX-MS). Other methods monitor the binding of an antibody to fragments of an antigen or mutated variations of an antigen, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered to be indicative of an epitope component. In addition, computational combinatorial methods can also be used for epitope mapping. These methods rely on the ability of the antibody of interest to select by affinity specific short peptides from combinatorial libraries of peptide phage displays. Antibodies having the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

Antibodies that compete with another antibody for binding to a target refer to antibodies that inhibit (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target and the extent to which one antibody inhibits the binding of another antibody to a target can be determined using known competition experiments. In certain embodiments, the antibody competes with another antibody and inhibits the binding of the other antibody to the target by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%. The level of inhibition or competition may vary depending on which antibody is the blocking antibody (ie, a cold antibody that is first incubated with the target). Competition analyzes can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101/pdb.prot4277 or in Chapter 11 Using Antibodies by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, overlapping epitope, or adjacent epitopes (e.g. as evidenced by steric mismatch).

Other competitive binding assays include direct or indirect radioimmunoassay (RIA), direct or indirect enzyme-linked immunosorbent assay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology, 9:242 (1983)); solid-phase direct EIA with biotin-avidin (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA assay using the 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotinavidin EIA assay (Cheung et al., Virology 176:546 (1990)) and direct label RIA assay (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment fusion. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term monoclonal antibody as used herein refers to an antibody that exhibits a single specificity and binding affinity for a particular epitope, or an antibody composition in which all antibodies exhibit a single specificity and binding affinity for a particular epitope. Accordingly, the term human monoclonal antibody refers to an antibody or antibody composition that exhibits a single binding specificity and that has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse, having a genome containing a human heavy chain transgene and a light chain transgene fused to immortalized cells.

The term recombinant human antibody as used herein includes all

- 18 043381 human antibodies produced, expressed, created or isolated by recombinant methods, such as (a) antibodies isolated from an animal (for example, mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or a hybridoma derived from them , (b) antibodies isolated from a host cell transformed to express an antibody, such as from a transfectome, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies produced, expressed, generated or isolated by any other by methods that involve splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences that are encoded by germline genes but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, for example, Lonberg (2005), Nature Biotech. 23(9):1117-1125), the variable region contains an antigen binding domain, which is encoded by various genes that are rearranged to form an antibody specific for foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple changes to a single amino acid (called somatic mutation or hypermutation) to increase the affinity of the antibody for a foreign antigen. The constant region will change with additional response to the antigen (i.e., isotype switching). Consequently, rearranged and somatically mutated nucleic acid molecules that encode immunoglobulin light chain and heavy chain polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecules, but will instead be substantially identical or similar (i.e., have at least 80% identity).

A human antibody (HuMAb) refers to an antibody having variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term human antibody, as used herein, is not intended to include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms human antibodies and fully human antibodies are used interchangeably.

A humanized antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of the antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, while some, most or all of the amino acids in one or more CDR regions are unchanged. Minor additions, deletions, insertions, substitutions or modifications of amino acids are acceptable as long as they do not abolish the ability of the antibody to bind to a specific antigen. The humanized antibody retains antigen specificity similar to that of the parent antibody.

A chimeric antibody refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a murine antibody and the constant regions are derived from a human antibody.

The term cross-reaction as used herein refers to the ability of an antibody described herein to bind to FAM19A5 from another species. For example, an antibody described herein that binds to human FAM19A5 may also bind to FAM19A5 from other species (eg, murine FAM19A5). As used herein, cross-reactivity can be measured by determining specific reactivity with purified antigen in binding assays (eg, SPR, ELISA) or binding to or otherwise functional interaction with cells physiologically expressing FAM19A5. Methods for determining cross-reactivity include standard binding assays as described herein, for example, using the BIACORE® surface plasmon resonance (SPR) assay using the BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometry methods.

The term naturally occurring as applied to an object in this document refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses), that can be isolated from a source in nature, and that has not been intentionally altered by a person in a laboratory is of natural origin.

A polypeptide refers to a chain containing at least two sequentially linked

- 19 043381 amino acid residues, without an upper limit on chain length. One or more amino acid residues in a protein may contain a modification such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A protein may contain one or more polypeptides.

The term nucleic acid molecule as used herein is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

The term vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a circular double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial replication origin and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). In general, expression vectors with useful properties in recombinant DNA techniques are often in the form of plasmids. In this description, plasmid and vector can be used interchangeably, since plasmid is the most widely used form of vector. However, other forms of expression vectors are also included, such as viral vectors (eg, replication-defective retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

The term recombinant host cell (or simply host cell) as used herein is intended to mean a cell that contains a nucleic acid not naturally present in the cell, and may be a cell into which a recombinant expression vector is inserted. It should be understood that such terms are intended to refer not only to a particular cell of a subject, but also to the progeny of such cell. Since certain modifications occur in subsequent generations due to mutations or environmental influences, such progeny may not be essentially identical to the parent cell, but are still included within the scope of the term host cell as used herein.

As used herein, the term bound refers to the connection of two or more molecules. The bond may be covalent or non-covalent. The relationship may also be genetic (ie, recombinant fusion). Such linkages can be achieved using a wide variety of methods known in the art, such as chemical conjugation and recombinant protein production.

As used herein, the term administration refers to the physical administration of a therapeutic agent or a composition containing a therapeutic agent to a subject using any of the various delivery methods and systems known to those skilled in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes, such as by injection or infusion.

The phrase parenteral administration as used herein means methods of administration other than enteral and local administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibody described herein may be administered by a non-parenteral route, such as the topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administration may also be performed, for example, once, multiple times and/or over one or more extended periods.

The terms treat, treatment and therapy as used herein refer to any type of intervention or process performed or active agent administered to a subject for the purpose of eliminating, alleviating, improving, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication , condition or biochemical features associated with the disease. The treatment may be treatment of a subject who has a disease or a subject who does not have a disease (eg, for prophylaxis).

As used herein, the term subject includes both human and non-human animals.

- 20 043381

The term non-human animal includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

As used herein, the term development of gliosis or development of reactive gliosis includes the onset or initiation of gliosis. Gliosis is a nonspecific reactive change in glial cells in the central nervous system (CNS, e.g., brain and/or spinal cord) in response to trauma or injury, e.g., trauma, spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune reactions and/or neurodegenerative diseases, and involves the proliferation or hypertrophy of several different types of glial cells, including astrocytes, microglia and oligodendrocytes. The development of gliosis can lead to the formation of scars that prevent axonal regeneration in the part of the central nervous system that has been injured or damaged. The detrimental effects of the development of gliosis include irreversible or permanent damage to neurons and/or prevention of recovery of surrounding neurons. Accordingly, the terms delay the development of gliosis and delay the development of reactive gliosis include inhibiting, delaying, suppressing or preventing the onset or initiation of gliosis and its associated detrimental effects on the CNS.

As used herein, the term overproliferation of reactive astrocytes includes an abnormal increase in the number of astrocytes due to destruction of adjacent neurons, for example, due to CNS injury, trauma, lesion, spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune responses and/or neurodegenerative diseases. Excessive proliferation of reactive astrocytes can lead to detrimental effects on the CNS, including scar formation that inhibits axonal regeneration in a portion of the CNS that has been injured or damaged, exacerbation of inflammation, production and release of neurotoxic levels of reactive oxygen species, release of potentially excitotoxic glutamate, potential contribution to pathogenesis of seizures, blood-brain barrier dysfunction, cytotoxic edema during trauma and stroke, potential activation of chronic cytokines by astrocytes contributing to chronic pain, and secondary degeneration after CNS injury. Sofroniew, Michael V. (2009), Trends in Neurosciences, 32(12):638-47; McGraw, J. et al. (2001), Journal of Neuroscience Research, 63(2):109-15; and Sofroniew, M.V. (2005), The Neuroscientist, 11(5):400-7. Accordingly, the term suppress excessive proliferation of reactive astrocytes includes inhibiting, slowing, suppressing, limiting or preventing excessive or abnormal proliferation of reactive astrocytes and the associated detrimental effects on the CNS.

As used herein, the term chondroitin sulfate proteoglycans includes proteoglycans consisting of a protein core and chondroitin sulfate. Chondroitin sulfate proteoglycans, also known as CSPGs, are extracellular matrix molecules widely expressed in the developing and adult CNS. CSGPs play a key role in neurodevelopment and glial scar formation, and they inhibit axonal regeneration after injury in the CNS. Notable CSPGs include aggrecan (CSPG1), versican (cSpG2), neurocan (CSPG3), CSPG4 (or neuronal glial antigen 2 (NG2)), CSPG5, SMC3 (CSPG6, chromosome structural maintenance 3), brevican (CSPG7) and CD44 (CSPG8, cluster of differentiation 44), phosphacaneurocan (CSPG3). Rhodes, K.E. and Fawcett, J.W. (2004), Journal of Anatomy. 204(1):33-48. Thus, the term reducing the expression of chondroitin sulfate proteoglycans includes reducing, inhibiting, reducing the level of one or more CSGPs or reducing the activity or rendering one or more CSGPs inactive. In some embodiments, the term includes reducing, inhibiting, reducing the level of neurocan, NG2, or both, or reducing the activity or rendering inactive neurocan, NG2, or both.

As used herein, the term neuron includes electrically excitable cells that process and transmit information through electrical and chemical signals. Neurons are the main components of the brain and spinal cord of the central nervous system and ganglia of the peripheral nervous system (PNS) and can connect with each other to form neural networks. A typical neuron consists of a cell body (soma), dendrites, and an axon. The soma (cell body) of a neuron contains the nucleus. The dendrites of a neuron are cellular extensions with many branches where most of the input information to the neuron originates. An axon is a thinner, cable-like extension that extends from the soma and carries nerve signals from the soma and some types of information back to the soma. The term promote neuronal regrowth includes stimulating, promoting, increasing or activating the growth of neurons, preferably after injury or damage.

As used herein, the term c-fos includes the proto-oncogene c-fos, which is rapidly induced by neurotransmitter stimulation. c-fos exists in many species, including mouse and human. The c-fos gene and protein are known and characterized. See Curran, T., The c-fos proto-oncogene, p. 307-327 (The Oncogene Handbook, Reddy EP et al. (eds.) Elsevier) (1988). The expression of c-fos can be determined by methods known in the art, for example Northern blotting, quantitative PCR or immunohistochemistry. The term increases c-fos expression includes an increase in mRNA levels

- 21 043381 c-fos, c-fos protein or c-fos protein activity.

As used herein, the term pERK includes phosphorylated extracellular signal-regulated kinase. Extracellular signal-regulated kinase or ERK, including ERK1 and ERK2, is a member of the mitogen-activated protein kinase (MAPK) family. ERK is activated by phosphorylation by its upstream kinase to form pERK, which then activates downstream targets. ERK is involved in neural and synaptic plasticity underlying learning, as well as memory and pain hypersensitivity. Ji R.R. et al., Nat. Neurosci. (1999), 2:1114-1119. The gene, protein, phosphorylation and activation of ERK are known and characterized, and the expression of ERK and pERK can be determined by methods known in the art (eg, Northern blotting, quantitative PCR or immunohistochemistry). See Gao Y.J. and Ji R.R., Open Pain J. (2009), 2:11-17. The term increased pERK expression includes increased levels of ERK mRNA, ERK protein, or ERK activity.

As used herein, GAP43, also known as growth-associated protein 43, is a neural tissue-specific protein that promotes neurite formation, regeneration, and plasticity. Benowitz L.I. and Routtenberg A. (1997), Trends in Neurosciences, 20(2):84-91; Aarts L.H. et al. (1998), Advances in Experimental Medicine and Biology, 446:85-106. Human GAP43 is encoded by the GAP43 gene. The polypeptide sequence of human GAP43 (UniProt: KB - PI7677) and the cDNA sequence encoding the polypeptide are known in the art. Kosik K.S. et al. (1988), Neuron, 1(2):127-32; Ng S.C. et al. (1988), Neuron, 1(2):133-9. Expression of GAP43 can be determined by methods known in the art (eg, Northern blotting, quantitative PCR or immunohistochemistry).

The term increase in GAP43 in neurons includes an increase or increase in the level of GAP43 mRNA, GAP43 protein, or increase in GAP43 protein activity.

As used herein, the term therapeutically effective amount refers to an amount of a drug, alone or in combination with another therapeutic agent, effective to treat a disease or disorder in a subject, or reduce the risk, potential, possibility, or occurrence of a disease or disorder (e.g., injury central nervous system). A therapeutically effective amount includes an amount of a drug or therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder (for example, damage to the central nervous system, such as traumatic brain injury or other disease described herein) . Thus, a therapeutically effective amount is an amount that reduces the risk, potential, likelihood or occurrence of a disease, or provides relief, mitigation of a disorder, or similar condition, and/or reduces at least one indicator (for example, the development of gliosis), and/or reduces at least one clinical symptom of a disease or disorder.

II. Antibodies to FAM19A5.

Disclosed herein are antibodies, such as monoclonal antibodies, that are characterized by certain functional features or properties. For example, antibodies specifically bind human FAM19A5, including soluble FAM19A5 and membrane-bound FAM19A5. In addition to specifically binding to soluble and/or membrane-bound FAM19A5, the antibodies described herein exhibit one or more of the following properties:

(a) reduce, eliminate, delay and/or prevent fibrosis;

(b) reduce the formation of excess extracellular matrix (ECM);

(c) delay tumor growth or progression;

(d) bind to soluble human FAM19A5 with a KD of 10 nM or less, as measured by enzyme-linked immunosorbent assay (ELISA);

(e) bind to membrane-bound human FAM19A5 with a K _D of 10 nM or less, as measured by ELISA;

(f) reduce, eliminate, delay and/or prevent the development of reactive gliosis;

(g) suppress excessive proliferation of reactive astrocytes;

(h) reduce the expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2);

(j) increase the expression of c-fos and pERK in the nucleus of neurons;

(k) promote neuronal survival;

(l) increase GAP43 expression in neurons;

(m) promote axon regrowth.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof specifically binds to soluble human or membrane-bound human FAM19A5 with high affinity, e.g., a K _D of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM ) or less, 10 ^-10 M (0.1 nM) or less, 10 ^-11 M or less, or 10 ^-12 M (1 pM) or less, for example,

- 22 043381

10-12-10 ^-7 M, 10-11-10 ^-7 M, 10 ^-10 -10 ^-7 M or 10 ^-9 -10 ^-7 M, for example, 10 ^-12 M, 5x10 ^-12 M, 10 ^{- 11} M, 5x10 ^-11 M, 10 ^-10 M, 5x10 ^-10 M, 10 ^-9 M, 5x10 ^-9 M, 10 ^-8 M, 5x10 ^-8 M, 10 ^-7 M or 5x10 ^-7 M. Standard tests to assess the ability of an antibody to bind to human FAM19A5 of various species are known in the art, including, for example, ELISA, Western blots and RIA. Suitable assays are described in detail in the Examples. The binding kinetics (eg, binding affinity) of antibodies can also be assessed using standard assays known in the art, such as ELISA, BIACORE® or KINEXA® assay. Assays to evaluate the effect of antibodies on the functional properties of FAM19A5 (eg, ligand binding) are described in more detail below and in the Examples. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to soluble human FAM19A5 with a KD, e.g., as determined by ELISA, 10 ^-7 M or less, 10 ^-8 M (10 nM) or less, 10 ^-9 M ( 1 nM) or less, 10 ^-10 M or less, 10 ^-12 -10 ^-7 M, 10 ^-11 -10 ^-7 M, 10 ^-10 -10 ^-7 M, 10 ^-9 -10 ^-7 M or 10 ^-8 -10 ^-7 M. In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof binds to soluble FAM19A5 with a KD of 10 nM or less, e.g., between 0.1 and 10 nM, between 0.1 and 5 nM, between 0 .1 and 1 nM, between 0.5 and 10 nM, between 0.5 and 5 nM, between 0.5 and 1 nM, between 1 and 10 nM, between 1 and 5 nM or between 5 and 10 nM. In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof specifically binds to soluble human FAM19A5 with a K _D of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 , 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 or 900 pM, or about 1, 2, 3, 4, 5, 6, 7, 8 or 9 nM, or about 10 , 20, 30, 40, 50, 60, 70, 80 or 90 nM, as determined by ELISA.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to membrane-bound human FAM19A5 with a K _D , e.g., as determined by ELISA, 10 ^-7 M or less, 10 ^-8 M (10 nM) or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, 10 ^-12 -10 ^-7 M, 10 ^-11 -10 ^-7 M, 10 ^-10 -10 ^-7 M, 10 ^-9 -10 ^-7 M or 10 ^-8 -10 ^-7 M. In certain embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof specifically binds to membrane-bound human FAM19A5 with a K _D of 10 nM or less, as determined by ELISA, e.g., between 0.1 and 10 nM, between 0.1 and 5 nM, between 0.1 and 1 nM, between 0.5 and 10 nM, between 0.5 and 5 nM, between 0.5 and 1 nM, between 1 and 10 nM, between 1 and 5 nM or between 5 and 10 nM. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to membrane-bound human FAM19A5 with a Kd of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70 , 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 or 900 pM, or about 1, 2, 3, 4, 5, 6, 7, 8 or 9 nM, or about 10, 20 , 30, 40, 50, 60, 70, 80 or 90 nM, as determined by ELISA.

The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can reduce, prevent, reverse, delay and/or inhibit the development of reactive gliosis, for example delay, slow or suppress non-specific reactive change of glial cells in the central nervous system (CNS, e.g. in the brain and/or or spinal cord) in response to injury or damage, for example due to trauma, spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune reactions and/or neurodegenerative diseases.

The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can delay, inhibit, retard, suppress, limit or prevent excessive or abnormal proliferation of reactive astrocytes and the associated detrimental effects on the CNS. For example, the anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention may inhibit or prevent the abnormal increase in astrocyte numbers due to neuronal destruction due to, for example, CNS injury, trauma, lesion, spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune responses and/or neurodegenerative diseases, inhibit or prevent scar formation in the CNS, inhibit or reduce the release of neurotoxic levels of reactive oxygen species or the release of potentially excitotoxic glutamate, reduce or inhibit seizures, pain and/or secondary degeneration after injury to the CNS. The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can promote, stimulate, increase or activate neuronal and/or axonal regrowth, preferably after trauma or injury to the CNS.

The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can inhibit the expression of chondroitin sulfate proteoglycans, including chondroitin sulfate core protein proteoglycans (CSGP), such as aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3), CSPG4 (or neuron -glial antigen 2 (NG2)), CSPG5, SMC3 (cSpG6, structural maintenance of chromosomes 3), brevican (CSPG7), CD44 (CSPG8, cluster of differentiation 44), phosphacaneurocan (CSPG3) or any combination thereof. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention inhibits, reduces, or reduces the level of Neurocan and/or NG2 or the activity of Neurocan and/or NG2.

The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can increase the expression of c-fos and pERK in neuronal nuclei, such as increasing the mRNA, protein and/or protein activity of c-fos and pERK. The anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention may also increase or enhance the expression level of GAP43 mRNA, GAP43 protein, or increase or enhance the activities of GAP43 protein.

In certain embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof of the present invention competes for binding to (or inhibits the binding of) a human FAM19A5 epitope with an anti-FAM19A5 antibody containing CDRs or variable regions described herein (e.g., 2-13, 3- 2 or 1-28). In certain embodiments, anti-FAM19A5 antibodies or antigen-binding portions thereof inhibit binding to a reference antibody comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein:

(i) The heavy chain CDR1, CDR2 and CDR3 of the reference antibody contain the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the light chain CDR1, CDR2 and CDR3 of the reference antibody contain the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 respectively;

(ii) heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 14, heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 15 and heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 16, light chain CDR1 contains the amino acid sequence of SEQ ID NO: 26 The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 27 and the light chain CDR3 contains the amino acid sequence of SEQ ID NO: 28; or (iii) the heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 20, the heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 21, the heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 22, the light chain CDR1 contains the amino acid sequence of SEQ ID NO: 32, light chain CDR2 contains the amino acid sequence of SEQ ID NO: 33 and light chain CDR3 contains the amino acid sequence of SEQ ID NO: 34.

In some embodiments, the reference antibody comprises (i) a heavy chain variable domain comprising SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 38, and (ii) a light chain variable domain comprising SEQ ID NO: 39. SEQ ID NO: 40 or SEQ ID NO: 42.

In certain embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof inhibits the binding of such reference antibody to human FAM19A5 by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%. Competing antibodies bind to the same epitope, overlapping epitope, or adjacent epitopes (eg, as evidenced by steric mismatch). Whether two antibodies compete with each other for binding to a target can be determined using competition experiments known in the art, such as RIA and EIA.

In certain embodiments, an anti-FAM19A5 antibody or antigen-binding portions thereof bind to the same FAM19A5 epitope as a reference antibody described herein comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein:

(i) Heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 11, Heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 12 and Heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 13, Light chain CDR1 contains the amino acid sequence of SEQ ID NO: 23 The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 24 and the light chain CDR3 contains the amino acid sequence of SEQ ID NO: 25;

(ii) heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 14, heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 15 and heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 16, light chain CDR1 contains the amino acid sequence of SEQ ID NO: 26 The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 27 and the light chain CDR3 contains the amino acid sequence of SEQ ID NO: 28; or (iii) wherein the heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 20, the heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 21, the heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 22, the light chain CDR1 contains the amino acid sequence of SEQ ID NO: : 32, light chain CDR2 contains the amino acid sequence of SEQ ID NO: 33 and light chain CDR3 contains the amino acid sequence of SEQ ID NO: 34.

In some embodiments, the reference antibody comprises (i) a heavy chain variable domain comprising SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 38, and (ii) a light chain variable domain comprising SEQ ID NO: 39 , SEQ ID NO: 40 or SEQ ID NO: 42.

Methods for determining whether two antibodies bind to the same epitope include, for example, epitope mapping techniques such as crystal X-ray crystal analysis of antigen:antibody complexes, which provides atomic resolution of the epitope, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). methods for monitoring the binding of an antibody to fragments of an antigen or mutated variations of an antigen, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered to be indicative of an epitope component,

- 24 043381 computational combinatorial methods for epitope mapping.

An anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention can bind to at least one epitope of mature human FAM19A5, as determined, for example, by antibody binding to fragments of human FAM19A5. In some embodiments, anti-FAM19A5 antibodies or antigen-binding portions thereof bind to at least one epitope that has the amino acid sequence TLDRDSSQPRRTIARQTARC (SEQ ID NO: 6 or amino acid residues 42-61 of SEQ ID NO: 2), or bind to a fragment located within the amino acid sequence of SEQ ID NO: 6, for example, an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids SEQ ID NO: 6. In some embodiments, antibodies to FAM19A5 or an antigen binding portion thereof bind to one or more amino acids corresponding to amino acid residues 46-51 (i.e., DSSQPR), such as amino acid residues 46 , 50 and 52 (i.e., D---P-R), such as amino acid residues 46, 47, 48, and 50 (i.e., DSS-P) SEQ ID NO: 2. In some embodiments, antibodies to FAM19A5 or antigen binding portions bind to a fragment located within the amino acid sequence of SEQ ID NO: 9, for example, an epitope having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19 or 20 amino acids SEQ ID NO: 9.

In some embodiments, the at least one epitope has an amino acid sequence that is at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 6. In some embodiments, the at least one epitope has an amino acid sequence that is at least 90% identical to 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 9.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds only to a human FAM19A5 epitope that is SEQ ID NO: 5, 6, 7, 8, 9, or 10, or a fragment located within the amino acid sequence of SEQ ID NO: 5. 6, 7, 8, 9 or 10, for example an epitope having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids SEQ ID NO: 5, 6, 7, 8, 9 or 10.

In some embodiments, an anti-FAM19A5 antibody or antigen binding portion thereof of the present invention is bound to SEQ ID NO: 6 or a fragment thereof in its native conformation (ie, not denatured). In some embodiments, an anti-FAM19A5 antibody or antigen binding portion thereof of the present invention is bound to SEQ ID NO: 9 or a fragment thereof in its native conformation (ie, not denatured). In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to both glycosylated and non-glycosylated human FAM19A5.

In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof is further bound to one or more additional epitopes of FAM19A5. Thus, certain anti-FAM19A5 antibodies or antigen-binding portions thereof bind to an epitope of SEQ ID NO: 6 and an additional epitope or an epitope of SEQ ID NO: 9 and an additional epitope. Other anti-FAM19A5 antibodies or antigen-binding portions thereof may bind to the epitope of SEQ ID NO: 5, SEQ ID NO: 9 and an additional epitope. In some embodiments, anti-FAM19A5 antibodies or antigen-binding portions thereof bind to an epitope of SEQ ID NO: 6, an epitope of SEQ ID NO: 10, and an additional epitope.

In some embodiments, one or more additional FAM19A5 epitopes is selected from QLAAGTCEIVTLDR (SEQ ID NO: 5, epitope F1), TLDRDSSQPRRTIARQTARC (SEQ ID NO: 6, epitope F2), TARCACRKGQIAGTTRARPA (SEQ ID NO: 7, epitope F3), ARPACVDARIIKTKQWCDML (SEQ ID NO: 8, epitope F4), CDMLPCLEGEGCDLINRSG (SEQ ID NO: 9, epitope F5) or NRSGWTCTQPGGRIKTTTVS (SEQ ID NO: 10, epitope F6) or a fragment located within the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or any combination thereof. A fragment located within the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 includes a fragment having 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of any SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, one or more additional FAM19A5 epitopes are selected from SEQ ID NO: 5, 6, 7, 8, 9, or 10 or a fragment located within the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9 or 10, for example a fragment having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids SEQ ID NO: 5, 6, 7, 8, 9 or 10, or any combination thereof.

In some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention binds to any one or more additional epitopes in

- 25 043381 their native conformation (i.e. not denatured). In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof binds to both glycosylated and non-glycosylated one or more additional epitopes of FAM19A5. In some embodiments, anti-FAM19A5 antibodies or antigen-binding portions thereof bind to at least one epitope of FAM19A5, defined as EP2, EP4, and/or EP8, wherein EP2 contains, is substantially composed of, or consists of DSSQP amino acids (SEQ ID NO: 66), wherein EP4 contains, substantially consists of, or consists of the amino acids ARCACRK (SEQ ID NO: 68) and wherein EP8 contains, substantially consists of, or consists of the amino acids TCTQPGGR (SEQ ID NO: 72). In some embodiments, the at least one epitope has an amino acid sequence that is at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to EP2, EP4 or EP8. In some embodiments, anti-FAM19A5 antibodies or an antigen-binding portion thereof bind only to EP2. In some embodiments, anti-FAM19A5 antibodies or an antigen-binding portion thereof bind to EP4 and EP8. In some embodiments, anti-FAM19A5 antibodies or an antigen-binding portion thereof bind to one or more FAM19A5 epitopes selected from the group consisting of SEQ ID NO: 65 [[EP1 - IVTLD]], SEQ ID NO: 67 [[EP3 - RTIAR] ], SEQ ID NO: 69 [[EP5 - ARPA]], SEQ ID NO: 70 [[EP6 - KTKQWCDML]], and SEQ ID NO: 71 [[EP7 - GCDLLINR]], and any combination thereof.

In certain embodiments, provided herein is an antibody or antigen-binding portion thereof that binds to FAM19A5 (e.g., human FAM19A5) with an affinity of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75. 80, 85, 90, 95% or higher than another FAM19A family protein, as measured by, for example, an immunoassay (eg, ELISA), surface plasmon resonance, or kinetic exclusion assay. In a specific embodiment, provided herein is an antibody or antigen-binding portion thereof that binds to FAM19A5 (eg, human FAM19A5) without cross-reactivity with another protein, as measured by, for example, an immunoassay.

In certain embodiments, anti-FAM19A5 antibodies are not native antibodies or naturally occurring antibodies. For example, antibodies to FAM19A5 have post-translational modifications that differ from naturally occurring antibodies, such as having more, less, or a different type of post-translational modification.

III. Typical antibodies to FAM19A5.

Specific antibodies that can be used in the methods described herein are antibodies, such as monoclonal antibodies, having the CDR and/or variable region sequences of antibodies 2-13, 3-2, 1-65 and 1-28 isolated in Example 1 and antibodies having at least 80% identity (eg, at least 85%, at least 90%, at least 95%, or at least 99% identity) to their variable region or CDR sequences. The amino acid sequences of VH 2-13, 3-2, 1-65 and 1-28 are presented in SEQ ID NO: 35-38. The amino acid sequences of VL 2-13, 3-2, 1-65 and 1-28 are presented in SEQ ID NO: 39-42.

table 2

Amino acid sequences of heavy chain variable CDRs (according to the Kabat system)

Antite LO VH- CDR1 VH-CDR2 VH-CDR3 Anti- FAM19 A5 (“213”) SHGMF (SEQ ID NO: 11) EITNDGSGTNYGS AVKG (SEQ ID NO: 12) STYECPGGFSCWGDTGQIDA (SEQ ID NO: 13) Anti- FAM19 A5 (“3-2”) SFNMF (SEQ ID NO: 14) QISSSGSSTNYAP AVRG (SEQ ID NO: 15) SSYDCPYGHCSSGVDSAGEIDA (SEQ ID NO: 16) Anti- FAM19 A5 ("1- 28") GFDFSD YG (SEQ ID NO: 20) IRSDGSNP (SEQ ID NO: 21) AKDGNGYCALDAYRSGGYSCG VYPGSIDA (SEQ ID NO: 22)

- 26 043381

Table 3

Amino acid sequences of light chain variable CDRs (according to the Kabat system)

Antibody VL-CDR1 VL-CDR2 VL-CDR3 Anti- FAM19A5 ("2-13") SGGSYSYG (SEQ ID NO: 23) WDDERPS (SEQ ID NO: 24) GTEDISGTAGV (SEQ ID NO: 25) Anti- FAM19A5 ("3-2") SGGGSYAGSYYYG (SEQ ID NO: 26) ESNKRPS (SEQ ID NO: 27) GSWDSSNGGI (SEQ ID NO: 28) Anti- FAM19A5 ("1-28") GYGYG (SEQ ID NO: 32) QND (SEQ ID NO: 33) GSEDSSTLAGI (SEQ ID NO: 34)

Table 4

Variable amino acid sequence of the heavy chain

Antibody Amino acid sequence of VH (SEQ ID NO) Anti- FAM19A 5 ("2-13") AVTLDESGGGLQTPGGALSLVCKASGFTFSSHGMFWVRQTP GKGLEYVAEITNDGSGTNYGSAVKGRATISRDNGQSTVRLQ LNNLRAEDTGTYFCARSTYECPGGFSCWGDTGQIDAWGHG TEVIVSS (SEQ ID NO: 35) Anti- FAM19A 5 AVTLDESGGGLQTPGGALSLVCKASGFTFSSFNMFWVRQAP GKGLEYVAQISSSGSSTNYAPAVRGRATISRDNGQSTVRLQL ("3-2") NNPGAEDTGTYYCAKSSYDCPYGHCSSGVDSAGEIDAWGH GTEVIVSS (SEQ ID NO: 36) Anti- FAM19A 5 ("1-28") AVTLDESGGGLQTPGGALSLVCKASGFDFSDYGMGWVRQA PGKGLEWVAAIRSDGSNPSYGSAVKGRATISKDNGRSTVRL QLNNLRAEDTATYYCAKDGNGYCALDAYRSGGYSCGVYPG SIDAWGHGTEVIVSS (SEQ ID NO: 38)

Table 5

Variable amino acid sequence of light chain

Antibody Amino acid sequence of VL (SEQ ID NO) Anti F.A. M19 A5 ("213") ALTQPSSVSANPGETVKITCSGGSYSYGWFQQKSPGSALVTVIYW DDERPSDIPSRFSGALSGSTNTLTITGVQADDEAVYFCGTEDISGTA GVFGAGTTLTVL (SEQ ID NO: 39) Anti FA M19 A5 ("32") ALTQPSSVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPV TLIYESNKRPSDIPSRFSGSTSGSTATLTITGVQADDEAIYYCGSWD SSNGGIFGAGTTLTVL (SEQ ID NO: 40) Anti F.A. ALTQPSSVSANLEGTVEITCSGSGYGYGWYQQKSPGSAPVTVIYQ NDKRPSDIPSRFSGSKSGSTGTLTITGVQVEDEAVYYCGSEDSSTLA GIFGAGTTLTVL (SEQ ID NO: 42) M19 A5 ("128")

- 27 043381

Accordingly, provided herein is an isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprising heavy and light chain variable regions, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, 36, or 38. In other embodiments, the isolated anti-FAM19A5 antibody or its antigen binding portion comprises a heavy chain variable region CDR selected from the group consisting of SEQ ID NO: 35, 36 or 38.

Also provided is an isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprising heavy and light chain variable regions, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39, 40, or 42. In other embodiments, the isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprises A light chain variable region CDR selected from the group consisting of SEQ ID NO: 39, 40, or 42. In certain embodiments, an isolated anti-FAM19A5 antibody or antigen binding portion thereof comprises a heavy chain variable region CDR selected from the group consisting of SEQ ID NO : 35, 36 or 38, and a light chain variable region CDR selected from the group consisting of SEQ ID NO: 39, 40 or 42.

Also provided is an isolated anti-FAM19A5 antibody or antigen binding portion thereof comprising heavy and light chain variable regions, wherein (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35 and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39; (ii) the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 40 or (iii) the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 38 and where the light chain variable region contains the amino acid sequence SEQ ID NO: 42.

Provided herein is an isolated anti-FAM19A5 antibody or antigen binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80% at least about 85 %, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or approximately 100% identical to the amino acid sequence shown as SEQ ID NO: 35, 36 or 38.

Also provided herein is an isolated anti-FAM19A5 antibody or antigen binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85% %, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or approximately 100% identical to the amino acid sequence shown as SEQ ID NO: 39, 40 or 42.

Also provided is an isolated anti-FAM19A5 antibody or antigen-binding portion thereof comprising heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical amino acid sequence represented by SEQ ID NO: 35, 36 or 38, and wherein the light chain variable region contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical in amino acid sequence, represented as SEQ ID NO: 39, 40 or 42.

In some embodiments, the present invention provides an isolated anti-FAM19A5 antibody or antigen-binding portion thereof, comprising:

(a) the sequences of the variable regions of the heavy and light chains containing SEQ ID NO: 35 and 39, respectively;

(b) sequences of the variable regions of the heavy and light chains containing

SEQ ID NO: 36 and 40 respectively; or (c) heavy and light chain variable region sequences containing

SEQ ID NO: 38 and 42 respectively.

In certain embodiments, an anti-FAM19A5 antibody or antigen-binding antibody thereof

- 28 043381 part of the present invention contains (i) heavy chain CDR1, CDR2 and CDR3 2-13 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 2-13 or any combinations thereof; (ii) heavy chain CDR1,

CDR2 and CDR3 3-2 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 3-2 or any combinations thereof or (iii) heavy chain CDR1, CDR2 and CDR3 1-28 or combinations thereof and/or light chain CDR1,

CDR2 and CDR3 1-28 or any combination thereof.

The amino acid sequences of VH CDR1, CDR2 and CDR3 for 2-13 are shown in SEQ ID NO: 11, 12 and 13, respectively. The amino acid sequences of VL CDR1, CDR2 and CDR3 for 2-13 are shown in SEQ ID NO: 23, 24 and 25, respectively. The amino acid sequences of VH CDR1, CDR2 and CDR3 for 3-2 are shown in SEQ ID NO: 14, 15 and 16, respectively. The amino acid sequences of VL CDR1, CDR2 and CDR3 for 3-2 are shown in SEQ ID NO: 26, 27 and 28, respectively. The amino acid sequences of VH CDR1, CDR2 and CDR3 for 1-28 are shown in SEQ ID NO: 20, 21 and 22, respectively. The amino acid sequences of VL CDR1, CDR2 and CDR3 for 1-28 are shown in SEQ ID NO: 32, 33 and 34, respectively. The amino acid sequences of VH CDR1, CDR2 and CDR3 for 1-65 are shown in SEQ ID NO: 17, 18 and 19, respectively. The amino acid sequences of VL CDR1, CDR2 and CDR3 for 1-65 are shown in SEQ ID NO: 29, 30 and 31, respectively.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention that specifically binds to human FAM19A5 comprises:

(a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 11; and/or (b) a VH CDR2 containing the amino acid sequence of SEQ ID NO: 12; and/or (c) a VH CDR3 containing the amino acid sequence of SEQ ID NO: 13.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VH CDRs identified above.

In some embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof that specifically binds to human FAM19A5 comprises:

(a) VL CDR1 containing the amino acid sequence of SEQ ID NO: 23; and/or (b) VL CDR2 containing the amino acid sequence of SEQ ID NO: 24; and/or (c) a VL CDR3 containing the amino acid sequence of SEQ ID NO: 25.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VL CDRs identified above.

In some embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof that specifically binds to human FAM19A5 comprises:

(a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 11;

(b) VH CDR2 containing the amino acid sequence of SEQ ID NO: 12;

(c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 13;

(d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 23;

(e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 24; and/or (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention that specifically binds to human FAM19A5 comprises:

(a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 14; and/or (b) a VH CDR2 containing the amino acid sequence of SEQ ID NO: 15; and/or (c) a VH CDR3 containing the amino acid sequence of SEQ ID NO: 16.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VH CDRs identified above.

In some embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof that specifically binds to human FAM19A5 comprises:

(a) VL CDR1 containing the amino acid sequence of SEQ ID NO: 26; and/or (b) VL CDR2 containing the amino acid sequence of SEQ ID NO: 27; and/or (c) a VL CDR3 containing the amino acid sequence of SEQ ID NO: 28.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VL CDRs identified above.

In some embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof that specifically binds to human FAM19A5 comprises:

(a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 14;

(b) VH CDR2 containing the amino acid sequence of SEQ ID NO: 15;

(c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 16;

(d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 26;

(e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 27; and/or (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 28.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VH CDRs identified above.

- 29 043381

In some embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof that specifically binds to human FAM19A5 comprises:

(a) VL CDR1 containing the amino acid sequence of SEQ ID NO: 29; and/or (b) VL CDR2 containing the amino acid sequence of SEQ ID NO: 30; and/or (c) a VL CDR3 containing the amino acid sequence of SEQ ID NO: 31.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VL CDRs identified above.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof of the present invention that specifically binds to human FAM19A5 comprises:

(a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 20; and/or (b) a VH CDR2 containing the amino acid sequence of SEQ ID NO: 21; and/or (c) a VH CDR3 containing the amino acid sequence of SEQ ID NO: 22.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VH CDRs identified above.

In some embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof that specifically binds to human FAM19A5 comprises:

(a) VL CDR1 containing the amino acid sequence of SEQ ID NO: 32; and/or (b) VL CDR2 containing the amino acid sequence of SEQ ID NO: 33; and/or (c) a VL CDR3 containing the amino acid sequence of SEQ ID NO: 34.

In specific embodiments, the antibody or antigen-binding portion thereof comprises one, two, or all three VL CDRs identified above.

In some embodiments, an anti-FAM19A5 antibody or an antigen-binding portion thereof that specifically binds to human FAM19A5 comprises:

(a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 20;

(b) VH CDR2 containing the amino acid sequence of SEQ ID NO:21;

(c) VH CDR3 containing the amino acid sequence of SEQ ID NO: 22;

(d) VL CDR1 containing the amino acid sequence of SEQ ID NO: 32;

(e) VL CDR2 containing the amino acid sequence of SEQ ID NO: 33; and/or (f) VL CDR3 containing the amino acid sequence of SEQ ID NO:34.

In specific embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof comprises one, two, three, four, five, or six CDRs as defined above.

A VH domain or one or more of its CDRs described herein may be linked to a constant domain to form a heavy chain, eg, a full-length heavy chain. Likewise, a VL domain or one or more of its CDRs described herein can be linked to a constant domain to form a light chain, eg, a full-length light chain. The full-length heavy chain and the full-length light chain combine to form the full-length antibody.

Accordingly, in specific embodiments, provided herein is an antibody comprising a light chain and a heavy chain of an antibody, such as a separate light chain and a heavy chain.

With respect to the light chain, in a particular embodiment, the light chain of the antibody described herein is a kappa light chain. In another specific embodiment, the light chain of an antibody described herein is a lambda light chain. In yet another specific embodiment, the light chain of an antibody described herein is a kappa light chain or a lambda light chain. In a specific embodiment, an antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain that contains any VL or VL CDR amino acid sequences described herein, and wherein the light chain constant region contains amino acid sequence of the human kappa light chain constant region. In a specific embodiment, an antibody described herein that specifically binds to a FAM19A5 polypeptide (e.g., human FAM19A5) comprises a light chain that contains the VL or VL CDR amino acid sequences described herein, and wherein the light chain constant region contains the amino acid sequence human lambda light chain constant region. Non-limiting examples of human constant region sequences have been described in the art, for example, see US Pat. No. 5,693,780 and Kabat E.A. et al. (1991) above.

With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In another specific embodiment, the heavy chain of the disclosed antibody may include a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In one embodiment, an antibody described herein that specifically binds

- 30 043381

FAM19A5 (eg, human FAM19A5), contains a heavy chain that contains a VH or VH CDR amino acid sequence described herein, and wherein the heavy chain constant region contains the amino acid sequence of a human gamma (γ) heavy chain constant region. In another embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a heavy chain that contains a VH or VH CDR amino acid sequence described herein, and wherein the heavy chain constant region comprises the amino acid sequence human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see US Pat. No. 5,693,780 and Kabat E.A. et al. (1991) above.

In some embodiments, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain containing a VH or VH CDR and a VL and VL CDR described herein, and wherein the constant regions contain the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another specific embodiment, an antibody described herein that specifically binds to FAM19A5 (e.g., human FAM19A5) comprises a VL domain and a VH domain comprising any of the amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions immunoglobulin molecules IgG, IgE, IgM, IgD, IgA or IgY, any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) immunoglobulin molecules. In some embodiments, the constant regions comprise the amino acid sequences of naturally occurring human IgG constant regions, including subclasses (eg, IgG1, IgG2, IgG3, or IgG4) and allotypes (eg, G1m, G2m, G3m, and nG4m) and variants thereof. See, for example, Vidarsson G. et al., Front Immunol. 5:520 (published online October 20, 2014) and Jefferis R. and Lefranc M.P., mAbs 1:4, 1-7(2009). In some embodiments, the constant regions comprise amino acid sequences of human IgG1, IgG2, IgG3, or IgG4 constant regions, or variants thereof.

In certain embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof described herein does not have Fc effector functions, such as complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated phagocytosis (ADCP). Effector functions are mediated by the Fc region and the residues most proximal to the hinge region in the CH2 domain of the Fc region are responsible for antibody effector functions because it contains a largely overlapping binding site for C1q (complement) and IgG Fc receptors (FcyR) on effector cells innate immune system. Also, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3 antibodies. Antibody effector functions can be reduced or avoided by various approaches known in the art, including (1) the use of antibody fragments lacking an Fc region (eg, such as Fab, F(ab') ₂ , single chain Fv (scFv) or sdAb consisting of a monomeric VH or VL domain); (2) generating aglycosylated antibodies, which can be produced, for example, by removing or altering the residue to which the sugar is attached, enzymatic removal of sugars, producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells unable to glycosylate proteins (eg, bacterial host cells, see, for example, US Patent Publication No. 20120100140); (3) the use of Fc regions from the IgG subclass that have reduced effector function (for example, an Fc region from IgG2 or IgG4 antibodies or a chimeric Fc region containing a CH2 domain from IgG2 or IgG4 antibodies, see, for example, US Publication No. 20120100140 and Lau S. et al., J. Immunol. 191:4769-4777 (2013)); and (4) obtaining an Fc region with mutations that result in reduced or absent Fc functions. See, for example, US Patent Publication No. 20120100140 and PCT US Patent Application cited therein, and An et al., mAbs 1:6, 572-579 (2009).

Thus, in some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof described herein is a Fab, Fab', F(ab') ₂ , Fv, single chain Fv (scFv), or sdAb consisting of a monomeric VH or VL domain . Such antibody fragments are well known in the art and are described above. In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof described herein comprises an Fc region with reduced or absent Fc function. In some embodiments, the constant regions comprise the amino acid sequences of the Fc region of human IgG2 or IgG4. In some embodiments, the anti-FAM19A5 antibody is an IgG2/IgG4 isotype. In some embodiments, the anti-FAM19A5 antibody comprises a chimeric Fc region that contains a CH2 domain from an IgG antibody of the IgG4 isotype and a CH3 domain from an IgG antibody of the IgG1 isotype, or a chimeric Fc region that contains a hinge region from IgG2 and a CH2 region from IgG4, or an Fc region with mutations that result in reduced or absent Fc functions. Fc regions with reduced Fc function or

- 31 043381 lacking it include those known in the art. See, for example, Lau S. et al., J.

Immunol. 191:4769-4777 (2013); An et al., mAbs 1:6, 572-579 (2009); and US Publication No. 20120100140, and US patents, publications, and PCT publications cited therein. Also Fc regions with reduced

Fc function or lack thereof can be easily manufactured by one skilled in the art.

IV. Nucleic acid molecules.

Another aspect described herein relates to one or more nucleic acid molecules that encode any one of the antibodies or antigen binding portions thereof described herein. Nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. A nucleic acid is isolated or rendered substantially pure when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA that is associated with isolated DNA in nature), or proteins by standard methods, including alkali/SDS treatment, CsCl separation, column chromatography, restriction enzymes, agarose gel electrophoresis and other methods well known in the art. See, F. Ausubel, et al., ed. (1987), Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid described herein may be, for example, DNA or RNA and may or may not contain intronic sequences. In some embodiments, the nucleic acid is DNA.

The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (eg, hybridomas obtained from transgenic mice having human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning methods. For antibodies obtained from an immunoglobulin gene library (eg, using phage display techniques), the nucleic acid encoding the antibody can be retrieved from the library.

The specific nucleic acid molecules described herein are molecules encoding the VH and VL sequences of monoclonal antibodies 2-13, 3-2, 1-65 and 1-28. Representative DNA sequences encoding the VH sequence 2-13, 3-2, 1-65 and 1-28 are given in SEQ ID NO: 43, 44, 45 and 46, respectively. Representative DNA sequences encoding VL sequences 2-13, 3-2, 1-65 and 1-28 are given in SEQ ID NO: 47, 48, 49 and 50, respectively.

Table 6

Heavy chain variable polynucleotide sequence

Antibody Amino acid sequence of VH (SEQ ID NO) Anti-FAM19A5 (2-13) GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGCCTC GTCTGCAAGGCCTCCGGGTTCACCTTCAGCAGCCATGGCATGTTCTGGGTGCGACAGACG CCCGGCAAGGGGTTGGAATATGTCGCTGAAATTACCAATGATGGTAGTGGCACAAACTAC GGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACAGT GAGG CTGCAGCTGAACAACCTCAGGGCTGAGGACACCGGCACCTACTTCTGCGCCAGATCTACT TATGAATGTCCTGGTGGTTTTAGTTGTTGGGGTGATACTGGTCAAATAGACGCATGGGGC CACGGGACCGAAGTCATCGTCTCCTCCA (SEQ ID NO: 43) Anti-FAM19A5 (3-2) GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGCCTC GTCTGCAAGGCCTCCGGGTTCACCTTCAGCAGCTTCAACATGTTCTGGGTGCGACAGGCG CCCGGCAAGGGGCTGGAATACGTCGCTCAAATTAGCAGCAGTGGTAGTAGCACAAACTAC GCACCCGCGGTGAGGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACAGT GAGG CTGCAGCTGAACAACCCCGGGGCTGAAGACACCGGCACCTACTACTGCGCCAAAAGTAGT TATGACTGTCCTTACGGTCATTGTAGTAGTGGTGTTGATAGTGCTGGTGAGATCGACGCA TGGGGCCACGGGACCGAAGTCATCGTCTCCTCCA (SEQ ID NO: 44) Anti-FAM19A5 (1-28) GCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGCCTC GTCTGCAAGGCCTCCGGGTTCGACTTCAGCGATTATGGCATGGGTTGGGTGCGACAGGCT CCAGGCAAGGGGCTGGAGTGGGTTGCTGCTATTAGAAGTGATGGTAGTAACCCATCATAC GGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAAGGACAACGGGCGAAGCACAGT GAGG CTGCAGCTGAACAACCTCAGGGCTGAGGACACCGCCACCTACTACTGCGCCAAGGATGGT AATGGTTACTGTGCTCTCGATGCTTATCGTAGTGGTGGTTATAGTTGTGGTGTTTATCCT GGTAGCATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 46)

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Table 7

Light chain variable polynucleotide sequence

Antibody Amino acid sequence of VL (SEQ ID NO) Anti-FAM19A5 (2-13) GGCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATAACCTG CTCCGGGGGTAGCTATAGCTATGGCTGGTTCCAGCAGAAGTCTCCTGGCAGTGCCCTTGT CACTGTGATCTACTGGGATGATGAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTGC CCTATCCGGCTCCACAAACACATTAACCATCACTGGGGTCCAAGCCGACGACGAGGCTG T CTATTTCTGTGGGACTGAAGACATCAGCGGCACTGCTGGTGTATTTGGGGCCGGGACAAC CCTGACCGTCCTGGG (SEQ ID NO: 47) Anti-FAM19A5 (3-2) GGCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTG CTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTACCAGCAGAAGGCACC TGGCAGTGCCCCTGTCACTCTGATCTATGAAAGCAACAAGAGACCCTCGGACATCCCTTC ACGATTCTCCGGTTCCACATCTGGCTCCACAGCCACACTAACCATCACTGGGGTCCAAGC CGATGACGAGGCTATCTATTACTGTGGGAGCTGGACAGTAGCAATGGTGGTATATTTGG GGCCGGGACAACCCTGACCGTCCTAGG (SEQ ID NO: 48) Anti-FAM19A5 (1-28) GCCCTGACTCAGCCGTCCTCGGTGTCAGCAAACCTGGAAGGAACCGTCGAGATCACCTGC TCCGGGAGTGGCTATGGTTATGGCTGGTATCAGCAGAAGTCTCCTGGCAGTGCCCCTGTC ACTGTGATCTATCAGAACGACAAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCC AAATCCGGCTCCACGGGCACATTAACCATCACTGGGGTCCAAGTCGAGGACGAGGCTG TC TATTACTGTGGGAGTGAAGACAGCAGCACTCTTGCTGGTATATTTGGGGCCGGGACAACC CTGACCGTCCTA (SEQ ID NO: 50)

The method for producing an anti-FAM19A5 antibody described herein may involve expressing the heavy chain and light chains in a cell line containing nucleotide sequences encoding the heavy and light chains with a signal peptide, for example, SEQ ID NO: 43 and 47, SEQ ID NO: : 44 and 48, SEQ ID NO: 46 and 50 respectively. Host cells containing these nucleotide sequences are included herein.

Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or an scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term operably linked, as used in this context, is intended to mean that two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in frame.

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions (hinge, CH1, CH2 and/or CH3). Human heavy chain constant region gene sequences known in the art (see, e.g., Kabat, E. A., et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained using standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, such as an IgG2 and/or an IgG4 constant region. For a heavy chain Fab gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a light chain Fab gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. Human light chain constant region gene sequences known in the art (see, e.g., Kabat, E. A., et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained using standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

To create the scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, for example encoding an amino acid sequence (Gly4-Ser3), so that the VH and VL sequences can be expressed as a contiguous single-stranded protein with the VL and VH connected by a flexible linker (see, for example, Bird et al. (1988), Science, 242:423-426; Huston et al. (1988), Proc. Natl. Acad. Sci. USA, 85:5879-5883 ; McCafferty et al. (1990), Nature, 348:552-554).

In some embodiments, the present invention provides a vector containing an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding portion thereof. In other embodiments, the vectors can be used for gene therapy.

Suitable vectors for the present invention include expression vectors, viral vectors and plasmid vectors. In one embodiment, the vector is a viral vector.

As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of the coding sequence of interest or, in the case of a viral RNA vector, the necessary elements for replication and translation, when introduced into an appropriate host cell . Expression vectors may include plasmids, phagemids, viruses and derivatives thereof.

Expression vectors of the present invention may include polynucleotides encoding an antibody or antigen-binding portion thereof described herein. In one embodiment, the coding sequences for an antibody or antigen-binding portion thereof are operably linked to an expression control sequence. As used herein, two nucleotide sequences are operably linked when they are covalently linked in such a way as to allow each constituent nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are so-called operably linked when they are covalently linked such that expression or transcription and/or translation of the coding sequence can be placed under the influence or control of the gene expression control sequence. Two DNA sequences are so-called functionally linked if induction of the gene expression sequence 5' by the promoter results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not prevent the ability of the promoter region to direct transcription of the coding sequence or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a gene expression sequence would be operably linked to a nucleic acid coding sequence if the gene expression sequence was capable of transcription of that nucleic acid coding sequence such that the resulting transcript was translated into the desired antibody or antigen binding portion thereof.

Viral vectors include, but are not limited to, the nucleic acid sequences of the following viruses: a retrovirus such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; viruses like SV40; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; cowpox virus; polio virus; and an RNA virus such as a retrovirus. Other vectors well known in the art can easily be used. Some viral vectors are based on noncytopathic eukaryotic viruses in which nonessential genes are replaced by the gene of interest. Noncytopathic viruses include retroviruses whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. The most useful retroviruses are those that have a replication defect (that is, they are able to direct the synthesis of desired proteins, but are not able to produce an infectious particle). Such genetically modified retroviral expression vectors are generally useful for highly efficient gene transduction in vivo. Standard protocols for producing replication-defective retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retroviruses with the packaging cell line, collecting viral particles from tissue culture media, and infecting target cells with viral particles) are presented in Kriegler. M., Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated virus can be engineered to be replication-deficient and capable of infecting a wide range of cell types and species. In addition, it has advantages such as resistance to heat and lipid solvents; high frequency of transduction in cells of different lines, including hematopoietic ones, and the absence of inhibition of superinfection, which allows for several series of transduction. According to recent data, an adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability in the expression of the integrated gene, characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been observed in tissue culture for over 100 passages in the absence of selective pressure, indicating that genomic integration of adeno-associated virus is a relatively stable event. Adeno-associated virus can also function extrachromosomally.

In other embodiments, the vector is derived from a lentivirus. In certain embodiments, the vector is a recombinant lentivirus vector capable of infecting non-dividing cells.

The lentiviral genome and proviral DNA typically have three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes

- 34 043381

RNA-directed DNA polymerase (reverse transcriptase), protease and integrase; and the env gene encodes viral envelope glycoproteins. 5' and 3' LTRs serve to stimulate transcription and polyadenylation of virion RNAs. The LTR contains all other cis-acting sequences required for viral replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and/or SIV).

Near the 5'-LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (Psi site). If the viral genome lacks the sequences required for encapsidation (or packaging of retroviral RNA into infectious virions), the cis defect prevents encapsidation of the genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins. The present invention provides a method for producing a recombinant lentivirus capable of infecting non-dividing cells, comprising transfecting a suitable host cell with two or more vectors carrying packaging functions, namely gag, pol and env, as well as rev and tat. As will be described below herein, vectors that lack a functional tat gene are desirable for certain applications. Thus, for example, a first vector may provide a nucleic acid encoding a viral gag gene and a viral pol gene, and another vector may provide a nucleic acid encoding a viral env gene to produce a packaging cell. Introduction of a vector providing a heterologous gene, referred to herein as a transfer vector, into this packaging cell produces a producer cell that releases infectious viral particles carrying the foreign gene of interest. According to the above configuration of vectors and foreign genes, the second vector may provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from virtually any suitable virus, including retroviruses. In some embodiments, the env protein is an amphotropic envelope protein that allows for transduction of cells from humans and other species.

Examples of env genes derived from retroviruses include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse mammary tumor virus (MuMTV or MMTV), gibbon monkey leukemia virus ( GaLV or GALV), human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes may also be used, such as vesicular stomatitis virus (VSV) G protein (VSV G), hepatitis and influenza virus. The vector providing the viral env nucleic acid sequence is operably linked to the regulatory sequences described elsewhere herein. In certain embodiments, the vector includes a lentiviral vector in which the HIV virulence genes env, vif, vpr, vpu and nef have been deleted without compromising the vector's ability to transduce non-dividing cells.

In some embodiments, the vector includes a lentiviral vector that contains a deletion of the U3 3'-LTR region. Deletion of the U3 region can be a complete or partial deletion. In some embodiments, a lentiviral vector of the present invention containing a FVIII nucleotide sequence described herein can be transfected into a cell with (a) a first nucleotide sequence comprising the gag, pol, or gag and pol genes, and (b) a second nucleotide sequence, containing a heterologous env gene; where the lentiviral vector lacks a functional tat gene. In other embodiments, the cell is further transfected with a fourth nucleotide sequence comprising the rev gene. In certain embodiments, the lentiviral vector lacks functional genes selected from vif, vpr, vpu, vpx and nef or a combination thereof. In some embodiments, the lentiviral vector contains one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polypurine tract (cPPT), or any combination thereof.

Examples of lentiviral vectors are described in WO 99/31251, WO 97/12622, WO 98/17815, WO 98/17816 and WO 98/18934, which are incorporated herein by reference in their entirety.

Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly useful for delivering genes to cells in vivo because their inability to replicate within the host genome and integrate into it. However, these plasmids, having a promoter compatible with the host cell, can express a peptide from a gene functionally encoded in the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40 and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, cat. no. V86320; and pBudCE4.1, part number V53220, all from Invitrogen (Carlsbad, CA). Other plasmids are well known to those skilled in the art. In addition, plasmids can be custom-made using standard molecular biology techniques to remove and/or add specific DNA fragments.

V. Obtaining antibodies.

Antibodies or fragments thereof that immunospecifically bind to FAM19A5 (eg, human FAM19A5) can be produced by any method known in the art for antibody synthesis, such as chemical synthesis or recombinant expression methods. The methods described herein employ, unless otherwise indicated, conventional techniques in the fields of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization and related fields. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, for example, Maniatis T. et al. (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J. et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J. et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel F.M. et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren V. et al. (eds.) (1999), Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

In a specific embodiment, an antibody described herein is an antibody (eg, a recombinant antibody) produced, expressed, created or isolated by any method that includes the creation, for example, by synthesis, genetic engineering, of DNA sequences. In certain embodiments, such an antibody contains sequences (eg, DNA sequences or amino acid sequences) that do not naturally exist in the germline repertoire of an animal or mammalian (eg, human) antibody in vivo.

In a certain aspect, provided herein is a method of producing an antibody or antigen binding fragment thereof that immunospecifically binds to FAM19A5 (eg, human FAM19A5), comprising culturing a cell or host cell as described herein. In a certain aspect, provided herein is a method of producing an antibody or antigen binding fragment thereof that immunospecifically binds to FAM19A5 (e.g., human FAM19A5), comprising expressing (e.g., recombinant expression) the antibody or antigen binding fragment thereof using a cell or host cell described in herein (eg, a cell or host cell containing polynucleotides encoding an antibody described herein). In a specific embodiment, the cell is an isolated cell. In a specific embodiment, exogenous polynucleotides have been introduced into the cell. In a specific embodiment, the method further includes the step of purifying an antibody or antigen binding fragment thereof obtained from a cell or host cell. Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology (2002), ^5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York). Monoclonal antibodies can be produced using a wide range of techniques known in the art, including the use of hybridoma technology, recombinant technology and phage display technology, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and described in, for example, Harlow E. & Lane D., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press , ^2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas, 563 681 (Elsevier, NY, 1981).

The term monoclonal antibody as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein, or a fragment thereof, such as the light chain and/or heavy chain of such an antibody.

In specific embodiments, a monoclonal antibody, as used herein, is an antibody produced by a single cell (e.g., a hybridoma or a host cell producing a recombinant antibody), wherein the antibody immunospecifically binds to FAM19A5 (e.g., human FAM19A5), as defined by, e.g. using an ELISA or other antigen binding or competitive binding assay known in the art or as exemplified herein. In specific embodiments, the monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent or multivalent (eg, bivalent) antibody. In specific embodiments, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). Monoclonal antibodies described herein can, for example, be produced by a hybridoma method, as described in Kohler G. & Milstein C. (1975), Nature, 256:495, or can be, for example, isolated

- 36 043381 lena from phage libraries, for example, using the methods described in this document. Other methods for producing clonal cell lines and the monoclonal antibodies they express are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology (2002), ^5th Ed., Ausubel FM et al., supra). Methods for producing and screening specific antibodies using hybridoma technology are conventional and well known in the art. For example, in a hybridoma method, a mouse or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster, or macaque, is immunized to identify lymphocytes that produce or are capable of producing antibodies that specifically bind to a protein (e.g., human FAM19A5) used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with the myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, p. 59-103 (Academic Press, 1986)). In addition, the RIMMS (repeated immunization multiplex) technique can be used to immunize an animal (Kilpatrick KE et al. (1997), Hybridoma, 16:381-9, incorporated by reference in its entirety). In some embodiments, mice (or other animals such as chickens, rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) may be immunized with an antigen (e.g., FAM19A5, such as human FAM19A5), and if an immune response is detected, for example, antigen-specific antibodies are detected in mouse serum, the mouse spleen is collected and splenocytes are isolated. The splenocytes are then fused by well-known methods with any suitable myeloma cells, such as the SP20 cell line available from the American Type Culture Collection (ATCC) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limited breeding. In some embodiments, lymph nodes from immunized mice are harvested and fused with NSO myeloma cells.

The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells. In particular embodiments, myeloma cells are used that efficiently fuse, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium, such as a HAT medium. Among these myeloma cell lines are murine myeloma lines such as NSO cell lines or mouse tumor-derived lines MOPC-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63, Ag8.653 cells, available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D. (1984), J. Immunol. 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, p. 51 -63 (Marcel Dekker, Inc., New York, 1987)).

The culture medium in which hybridoma cells grow is assayed for the production of monoclonal antibodies directed to FAM19A5 (eg, human FAM19A5). The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Once hybridoma cells that produce antibodies of the desired specificity, affinity and/or activity have been identified, clones can be subcloned by limiting dilution procedures and grown using standard methods (Goding J.W. (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum using conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. The antibodies described herein include antibody fragments that recognize specific FAM19A5 (eg, human FAM19A5) and can be prepared by any method known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of two identical arms of the antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds at the hinge region.

In addition, the antibodies described herein, or antigen-binding fragments thereof, can also be produced using various phage display methods known in the art.

- 37 043381 new field of technology. In phage display methods, the functional domains of antibodies are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, from mouse or chicken cDNA libraries of human or non-human diseased tissues). The DNAs encoding the VH and VL domains are recombined together with the scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and E. coli is infected with a helper phage. The phage used in these methods is usually filamentous phage, including fd and M13, and the VH and VL domains are usually recombinantly fused to the phage III gene or VIII gene. A phage expressing an antigen-binding domain that binds to a specific antigen can be selected for or identified with the antigen, for example, using a labeled antigen or an antigen bound or captured on a solid surface or bead. Examples of phage display methods that can be used to produce the antibodies described herein include those described in Brinkman U. et al. (1995), J. Immunol. Methods, 182:41-50; Ames R.S. et al. (1995), J. Immunol. Methods, 184:177-186; Kettleborough C.A. et al. (1994), Eur. J. Immunol. 24:952-958; Persic L. et al. (1997), Gene 187:9-18; Burton D.R. & Barbas C.F. (1994), Advan. Immunol. 57:191-280; PCT Publication No. PCT/GB91/001134; international publication No. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401 and WO 97/13844 and US patent No. 5698426, 5223409 , 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743 and 5969108.

As described in the above references, following phage selection, antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies or any other desired antigen binding moiety, and expressed in any desired host, including mammalian cells, insect cells, plants, yeasts and bacteria, for example as described below. Methods for recombinant production of antibody fragments such as Fab, Fab' and F(ab') ₂ fragments can also be used using methods known in the art, such as those described in PCT Publication No. WO 92/22324; Mullinax RL et al. (1992), BioTechniques, 12(6):864-9; Sawai H. et al. (1995), Am. J. Reprod. Immunol. 34:26-34 and Better M. et al. (1988), Science, 240:1041-1043.

In one aspect, PCR primers comprising VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to generate whole antibodies to amplify VH or VL sequences from a template, such as scFv clones. Using cloning techniques known to those skilled in the art, PCR amplified VH domains can be cloned into vectors expressing the VH constant region, and PCR amplified VL domains can be cloned into vectors expressing the VL constant region, such as human constant region kappa or lambda regions. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vectors and the light chain conversion vectors are then co-transfected into cell lines to create stable or transient cell lines that express full-length antibodies, such as IgG, using methods known to those skilled in the art.

A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a monoclonal antibody from a non-human animal (eg, mouse, rat or chicken) fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison S.L. (1985), Science, 229:1202-7; Oi V.T. & Morrison S.L. (1986), BioTechniques, 4:214-221; Gillies S.D. et al. (1989), J. Immunol. Methods, 125:191-202 and US Patent Nos. 5807715, 4816567, 4816397 and 6331415.

The humanized antibody is capable of binding to a predetermined antigen and contains a framework region having essentially the amino acid sequence of a human immunoglobulin, and CDRs having essentially the amino acid sequence of a non-human immunoglobulin (eg, mouse or chicken immunoglobulin). In specific embodiments, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically one that is part of a human immunoglobulin. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 heavy chain regions. The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Humanized antibodies can be prepared using a variety of methods known in the art, including, but not limited to, CDR coupling (EP No. EP 239400; International Publication No. WO 91/09967 and US Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veining or modification of the surface (European patents No. EP 592106 and EP 519596; Padlan E.A. (1991), Mol. Immunol. 28(4/5):489-498; Studnicka G.M. et al. (1994), Prot. Engineering, 7(6 ):805-814 and Roguska M.A. et al. (1994), PNAS, 91:969-973), strand shuffling (U.S. Patent No. 5,565,332) and methods described, for example, in U.S. Patent Nos. 6,407,213, 5,766,886, int.

- 38 043381 native publication No. WO 93/17105; Tan P. et al. (2002), J. Immunol. 169:1119-25; Caldas S. et al. (2000), Protein Eng. 13(5):353-60; Morea V. et al. (2000), Methods, 20(3):267-79; Baca M. et al. (1997), J. Biol. Chem. 272(16):10678-84; Roguska M.A. et al. (1996), Protein Eng. 9(10):895 904; Couto J.R. et al. (1995), Cancer Res. 55(23 Supp):5973-5977; Couto J.R. et al. (1995), Cancer Res. 55(8):1717-22; Sandhu J.S. (1994), Gene 150(2):409-10 and Pedersen J.T. et al. (1994), J. Mol. Biol. 235(3):959-73. See also US Application Publication No. 2005/0042664 A1 (Feb. 24, 2005), which is incorporated herein by reference in its entirety.

Methods for producing multispecific (eg, bispecific) antibodies have been described. See, for example, US Pat. Nos. 7,951,917; 7,183,076; 8,227,577; 5837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713. Single domain antibodies, such as antibodies lacking light chains, can be produced by methods well known in the art.See Riechmann L. & Muyldermans S. (1999), J. Immunol. 231:25-38; Nuttall S. D. et al. (2000), Curr Pharm. Biotechnol. 1(3): 253-263; Muyldermans S., (2001), J. Biotechnol. 74(4):277-302; US patent No. 6005079 and international publications No. WO 94/04678, WO 94/25591 and WO 01/44301. In addition , antibodies that immunospecifically bind to the FAM19A5 antigen can in turn be used to generate anti-idiotypic antibodies that mimic the antigen using methods well known to those skilled in the art (See, for example, Greenspan N.S. & Bona S.A. (1989), FASEB J. 7(5):437-444 and Nissinoff A. (1991), J. Immunol. 147(8):2429-2438).

In specific embodiments, an antibody described herein that binds to the same FAM19A5 epitope (eg, human FAM19A5) as an anti-FAM19A5 antibody described herein is a human antibody or an antigen binding fragment thereof. In specific embodiments, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) antibodies described herein (e.g., 2-13 and 3-2) from binding to FAM19A5 (e.g., human FAM19A5) , is a human antibody or an antigen-binding fragment thereof.

Human antibodies can be obtained using any method known in the art. For example, it is possible to use transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes. In particular, gene complexes of the heavy and light chains of human immunoglobulins can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse immunoglobulin heavy and light chain genes can become nonfunctional individually or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are routinely immunized with a selected antigen, eg all or part of an antigen (eg FAM19A5). Monoclonal antibodies directed against an antigen can be generated from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutations. Thus, using this technique, therapeutically useful IgG, IgA, IgM and IgE antibodies can be obtained. For a review of this technology for the production of human antibodies, see Lonberg N. & Huszar D. (1995), Int. Rev. Immunol. 13:65-93. For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, International Publications No. WO 98/24893, WO 96/34096 and WO 96/33735 and US Patent Nos. 5413923, 5625126, 5633425, 5569825, 5661016, 5545806, 5814318 and 5939598. Examples of mice capable of producing human antibodies include XENOMOUSE™ (Abgenix, Inc.; US Patent Nos. 6075181 and 6150184), HUAB-MOUSE™ (Mederex, Inc./Gen Pharm; patent s USA No. 5545806 and 5569825), TRANS CHROMO MOUSE™ (Kirin) and KM MOUSE™ (Medarex/Kirin).

Human antibodies that specifically bind to FAM19A5 (eg, human FAM19A5) can be produced by various methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also US Patent Nos. 4,444,887, 4,716,111 and 5,885,793 and International Publications Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes are transformed

- 39 043381 baths of Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened for those that secrete human monoclonal antibodies that immunospecifically bind to the target antigen (eg, FAM19A5, such as human FAM19A5)). Such methods are known and described in the art, see, for example, Shinmoto H. et al. (2004), Cytotechnology, 46:19-23; Naganawa Y. et al. (2005), Human Antibodies, 14:27-31.

VI. Methods for constructing antibodies.

As discussed above, an anti-FAM19A5 antibody or an antigen-binding portion thereof having the VH and VL sequences described herein can be used to create a new anti-FAM19A5 antibody or an antigen-binding portion thereof by modifying the VH and/or VL sequences or constant region(s) attached to it. Thus, in another aspect described herein, structural features of an anti-FAM19A5 antibody described herein, such as 2-13 and 3-2, are used to generate structurally related anti-FAM19A5 antibodies that retain at least one functional property of the antibodies , described herein, such as binding to human FAM19A5. For example, the starting material for the design method is the VH and/or VL sequences provided herein, or one or more CDR regions. To create an engineered antibody, it is not necessary to actually produce (ie, express as a protein) an antibody having one or more VH and/or VL sequences presented herein, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as starting material to create second-generation sequence(s) derived from the original sequence(s), and the second-generation sequence(s) are then prepared and expressed as a protein .

Accordingly, provided herein are methods of producing an antibody to FAM19A5 or an antigen-binding portion thereof, comprising:

(a) providing (i) a heavy chain variable region sequence containing the sequence of CDR1, CDR2 and/or CDR3, as indicated in table. 2, or CDR1, CDR2 and/or CDR3 of the heavy chain variable region, as indicated in table. 4; and (ii) a light chain variable region sequence containing the sequence of CDR1, CDR2 and/or CDR3, as indicated in table. 3, or CDR1, CDR2 and/or CDR3 of the light chain variable region, as indicated in table. 5;

(b) altering at least one amino acid residue in the heavy chain variable region sequence and/or the light chain variable region sequence to create at least one altered antibody sequence or antigen binding portion; and (c) expressing the altered antibody sequence or antigen-binding portion as a protein.

Standard molecular biology techniques can be used to obtain and express the altered antibody sequence or antigen-binding portion thereof.

In some embodiments, the antibody or antigen-binding portion thereof encoded by the altered antibody or antigen-binding portion sequence(s) is a sequence that retains one, some, or all of the functional properties of the anti-FAM19A5 antibodies described herein, which mean that the antibody:

(1) binds to soluble human FAM19A5, for example, with a KD of 10 nM or less (eg, 0.01-10 nM), for example, as measured by Biacore;

(2) binds to membrane-bound human FAM19A5, for example, with a KD of 1 nM or less (eg, 0.01-1 nM), for example, as measured by ELISA;

(3) binds to membrane-bound human FAM19A5, for example, with an EC ₅₀ of 1 nM or less (eg, 0.01-1 nM), for example, as measured by ELISA;

(4) reduces, eliminates, delays and/or prevents the development of reactive gliosis;

(5) suppresses excessive proliferation of reactive astrocytes;

(6) reduces the expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2);

(7) increases the expression of c-fos and pERK in the nucleus of neurons;

(8) promotes neuronal survival;

(9) increases GAP43 expression in neurons;

(10) promotes axon regrowth;

(11) competes in either or both directions for binding to human FAM19A5 with 2-13, 3-2, or 1-28.

The altered antibody or antigen-binding portion thereof may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or all functional properties listed above as (1)-(11). The functional properties of altered antibodies or their antigen-binding parts may be

- 40 043381 were evaluated using standard assays available in the art and/or described herein, such as those set forth in the examples (eg, ELISA, FACS).

In certain embodiments of the antibody engineering methods described herein, mutations may be introduced randomly or selectively throughout all or part of the anti-FAM19A5 antibody coding sequence, and the resulting modified anti-FAM19A5 antibodies may be screened for binding activity and/or other functional properties as described in this document. Mutation methods have been described in the art. For example, PCT Publication No. WO 02/092780 from Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication No. WO 03/074679 from Lazar et al. methods for using computer screening methods to optimize the physicochemical properties of antibodies are described.

VII. Cells and vectors.

In certain aspects, provided herein are cells (e.g., host cells) expressing (e.g., recombinantly) antibodies described herein (or an antigen binding fragment thereof) that specifically bind to FAM19A5 (e.g., human FAM19A5) and related polynucleotides, and expression vectors. Provided herein are vectors (eg, expression vectors) containing polynucleotides containing nucleotide sequences encoding anti-FAM19A5 antibodies or a fragment for recombinant expression in host cells, such as mammalian cells. Also provided herein are host cells containing such vectors for recombinant expression of the anti-FAM19A5 antibodies described herein (eg, a human or humanized antibody). In a specific aspect, provided herein are methods of producing an antibody described herein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., a full-length antibody, a heavy and/or light chain antibody, or a single chain antibody described herein) that specifically binds to FAM19A5 (e.g., human FAM19A5) involves constructing an expression vector, containing a polynucleotide encoding an antibody. Once a polynucleotide encoding an antibody molecule, an antibody heavy and/or light chain, or a fragment thereof (e.g., heavy and/or light chain variable domains) described herein is obtained, a vector for producing the antibody molecule can be produced using recombinant DNA technology using methods well known in the art. Thus, methods for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antibody fragment (eg, a light chain or a heavy chain) are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding an antibody or antibody fragment (eg, light chain or heavy chain) and corresponding transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA methods, in vivo synthesis methods, and genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, a heavy or light chain variable domain of an antibody or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may, for example, include a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, International Publications Nos. WO 86/05807 and WO 89/01036 and US Pat. No. 5,122,464), and the variable domains of the antibody can be cloned into such a vector to express the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

The expression vector can be transferred into a cell (eg, a host cell) by conventional methods, and the resulting cells can then be cultured by conventional methods to produce the antibody described herein (eg, an antibody containing VH and/or VL, or one or several CDRs VH and/or VL, 2-13, 3-2 or 1-28), or a fragment thereof. Thus, provided herein are host cells containing a polynucleotide encoding an antibody described herein, or fragments thereof, or a heavy or light chain, or fragment thereof, or a single chain antibody described herein, operably linked to a promoter to express such sequences in the host cell. In certain embodiments, for the expression of double chain antibodies, vectors encoding both the heavy and light chains separately can be co-expressed in a host cell to express the entire immunoglobulin molecule, as described in detail below. In certain embodiments, the host cell contains a vector containing a polynucleotide encoding both a heavy chain and a light chain of an antibody described herein, or a fragment thereof. In specific embodiments, the host cell contains two different vectors: a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and a second vector containing a polynucleotide encoding the light chain or a light chain variable region of an antibody described herein, or a fragment thereof. In other embodiments, the first host cell contains a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein, or a fragment thereof, and the second host cell contains a second vector containing a polynucleotide encoding the light chain or the light chain variable region of an antibody described herein. In specific embodiments, the heavy chain/heavy chain variable region is expressed by a first cell associated with the light chain/light chain variable region of a second cell to form the anti-FAM19A5 antibody described herein, or an antigen binding fragment thereof. In certain embodiments, a population of host cells is provided herein, including such a first host cell and such a second host cell.

In a specific embodiment, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain/light chain variable region of an anti-FAM19A5 antibody described herein and a second vector comprising a polynucleotide encoding a heavy chain/heavy chain variable region antibodies to FAM19A5 described in this document. A variety of host expression vector systems can be used to express the antibody molecules described herein. Such host expression systems are vehicles through which coding sequences of interest can be produced and subsequently purified, but are also cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule described herein in situ. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg, Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems (eg, green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmids) containing antibody coding sequences; or mammalian cell systems (e.g. COS (e.g. COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa and NIH 3T3, HEK293T, HepG2, SP210 R1, BW , LM, BSC1, BSC40, YB/20 and BMT10), carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, the late adenovirus promoter, the vaccinia virus 7.5K promoter ). In a specific embodiment, the cells for expressing the antibodies described herein or the antigen binding fragment are CHO cells, for example CHO cells from the CHO GS SYSTEM™ (Lonza). In a specific embodiment, the antibody expression cells described herein are human cells, such as human cell lines. In a specific embodiment, the mammalian expression vector is POPTIVEC™ or pcDNA3.3. In a specific embodiment, bacterial cells, such as Escherichia coli, or eukaryotic cells (eg, mammalian cells), especially for expressing the entire recombinant antibody molecule, are used to express the recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with a vector, such as the core intermediate early gene promoter element from human cytomegalovirus, are an effective antibody expression system (Foecking M.K. & Hofstetter H. (1986), Gene, 45:101-5 and Cockett M. I. et al (1990), Biotechnology, 8(7):662-7). In certain embodiments, the antibodies described herein are produced by CHO cells or NSO cells. In a specific embodiment, the expression of nucleotide sequences described herein encoding antibodies that immunospecifically bind FAM19A5 (eg, human FAM19A5) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, a number of expression vectors may advantageously be selected depending on the intended use of the antibody molecule being expressed. For example, when large quantities of such an antibody are needed to create pharmaceutical compositions of the antibody molecule, vectors that drive the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U. & Mueller-Hill B. (1983), EMBO J. 2:1791-1794), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is formed; pIN vectors (Inouye S. & Inouye M.

- 42 043381 (1985), Nuc. Acids Res. 13:3101-3109; Van Heeke G. & Schuster S.M. (1989), J. Biol. Chem. 24:5503-5509); and so on. For example, pGEX vectors can also be used to express foreign polypeptides as glutathione 5-transferase (GST) fusion proteins. Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose matrix beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST fragment. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector for the expression of foreign genes. The virus grows in Spodoptera fragiperda cells. The antibody coding sequence can be individually cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

A number of viral expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to the adenovirus transcription/translation control complex, such as a late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg, the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (eg, see Logan J. & Shenk T. (1984). PNAS, 81 (12):3655-9). Specific initiation signals may also be required for efficient translation of integrated antibody coding sequences. These signals include the ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to allow translation of the entire insert. These exogenous translational control signals and start codons can have various origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (See, for example, Bitter G. et al. (1987), Methods Enzymol. 153:516-544). In addition, a host cell strain may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a particular desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery to properly process the primary transcript, glycosylate, and phosphorylate the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK,

HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that non-endogenously produces any immunoglobulin chains), CRL7030, COS (e.g. COS 1 or COS), PER.C6, VERO, HsS78Bst, HEK293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells. In certain embodiments, the anti-FAM19A5 antibodies described herein are produced in mammalian cells, such as CHO cells. In a specific embodiment, the antibodies described herein, or antigen-binding portions thereof, have reduced fucose content or no fucose at all. Such antibodies can be prepared using techniques known to one skilled in the art. For example, antibodies may be expressed in cells deficient in or lacking the ability to fucosylate. In a specific example, cell lines with both alleles of 1,6-fucosyltransferase knocked out can be used to produce antibodies or antigen-binding portions thereof with reduced fucose content. The POTELLIGENT® system (Lonza) is an example of such a system that can be used to produce antibodies or antigen-binding portions thereof with reduced fucose content.

For long-term production of recombinant proteins with high yield, stable expression cells can be obtained. For example, cell lines can be constructed that stably express the anti-FAM19A5 antibody described herein, its antigen-binding portion. In specific embodiments, a cell provided herein stably expresses a light chain/light chain variable domain and a heavy chain/heavy chain variable domain that bind to form an antibody described herein, or an antigen binding portion thereof.

In certain aspects, instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by the

- 43 043381 appropriate expression control elements (eg promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After the introduction of foreign DNA/polynucleotide, the engineered cells can grow for 1-2 days in an enriched medium and then switch to a selective medium. A selectable marker in a recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method can advantageously be used to construct cell lines that express the anti-FAM19A5 antibody described herein or an antigen-binding portion thereof. Such engineered cell lines may be particularly useful in screening and evaluating compositions that directly or indirectly interact with an antibody molecule.

A number of selection systems can be used, including, but not limited to, thymidine kinase genes (Wigler M. et al. (1977), Cell, 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase genes (Szybalska E.H. & Szybalski W. (1962 ), PNAS, 48(12):2026-2034) and herpes simplex virus adenine phosphoribosyltransferase (Lowy I. et al. (1980), Cell, 22(3):817-23), which may be involved in tk-, hgprt - or aprt cells, respectively. In addition, antimetabolite resistance can be used as a basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler M. et al. (1980), PNAS, 77(6):3567-70; O'Hare K et al (1981), PNAS, 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan R.C. & Berg P. (1981), PNAS, 78(4):2072-6); neo, which confers resistance to aminoglycoside G-418 (Wu G.Y. & Wu C.H. (1991), Biotherapy, 3:87-95; Tolstoshev P. (1993), Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan R. C. (1993), Science, 260:926-932 and Morgan R. A. & Anderson W. F. (1993), Ann. Rev. Biochem. 62:191-217; Nabel G. J. & Feigner P. L. (1993), Trends Biotechnol. 11(5) :211-5); and hygro, which confers resistance to hygromycin (Santerre R.F. et al. (1984), Gene, 30(1-3):147-56).

Methods generally known in the art of recombinant DNA technology can generally be used to select the desired recombinant clone, and such methods are described, for example, in Ausubel F.M. et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M., Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in chapters 12 and 13, Dracopoli N.C. et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F. et al. (1981), J. Mol. Biol. 150:1-14, which are incorporated by reference herein in their entirety. Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington C.R. & Hentschel C.C.G., The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)).

When a marker in a vector system expressing an antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also be increased (Crouse G.F. et al. (1983), Mol. Cell Biol. 3:257-66).

A host cell can be co-transfected with two or more expression vectors described herein: a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors may contain identical selectable markers that provide equal expression of the heavy and light chain polypeptides. Host cells can be co-transfected with varying amounts of two or more expression vectors. For example, host cells can be transfected with any of the following ratios of the first expression vector and the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1: 8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

Alternatively, a single vector may be used that encodes and is capable of expressing both heavy and light chain polypeptides. In such situations, the light chain must be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot N.J. (1986), Nature, 322:562-565 and Kohler G. (1980), PNAS, 77:2197-2199). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. A multistroy nucleic acid construct may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotide sequences. For example, a bicistronic nucleic acid construct may comprise, in the order of a promoter, a first gene (eg, the heavy chain of an antibody described herein) and a second gene (eg, a light chain of an antibody described herein). In such an expression vector, the transcription of both genes can be driven by a promoter, while the translation of the mRNA from the first gene can be carried out by a cap-dependent scanning mechanism, and the translation of the mRNA from the second gene can be carried out by a cap-independent mechanism, such as IRES. Once an antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art to purify an immunoglobulin molecule, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen downstream of a protein A, and column chromatography), centrifugation, differential solubility, or any other standard method for protein purification. Additionally, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or other methods known in the art to facilitate purification.

In specific embodiments, an antibody or antigen-binding portion thereof described herein is isolated or purified. Typically, an isolated antibody is an antibody that contains substantially no other antibodies with antigenic specificity other than the isolated antibody. For example, in a particular embodiment, the antibody preparation described herein contains substantially no cellular material and/or chemical precursors. Formulations substantially free of cellular material include antibody preparations in which the antibody is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes antibody preparations having less than about 30, 20, 10, 5, 2, 1, 0.5, or 0.1% (by dry weight) of heterologous protein (also called herein, a contaminant protein) and/or antibody variants, such as various post-translationally modified forms of the antibody or other various antibody variants (or antigen binding portions). When an antibody is produced recombinantly, it also usually contains virtually no culture medium, i.e. the culture medium is less than about 20, 10, 2, 1, 0.5, or 0.1% by volume of the protein preparation. When an antibody is produced by chemical synthesis, it typically contains virtually no chemical precursors or other chemicals, i.e. it is separated from chemical precursors or other chemicals that are involved in protein synthesis. Accordingly, such antibody preparations contain less than about 30, 20, 10, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, the antibodies described herein are isolated or purified.

VIII. Analyzes.

The antibodies described herein can be tested for binding to FAM19A5 using, for example, a standard ELISA. Briefly, microtiter plates were coated with purified FAM19A5 at a concentration of 1–2 μg/ml in PBS and then blocked with 5% bovine serum albumin in PBS. Antibody dilutions (eg, dilutions of plasma from mice immunized with FAM19A5) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS/Tween and then incubated with a secondary reagent (eg, for human antibodies, goat-anti-human IgG Fc-specific polyclonal reagent) conjugated to horseradish peroxidase (HRP) for 1 hour at 37°C. After washing, the plates are treated with ABTS substrate (Moss Inc, product: ABTS-1000) and analyzed using a spectrophotometer at OD 415-495. Sera from immunized mice are then further screened by flow cytometry for binding to a cell line expressing human FAM19A5, but not to a control cell line that does not express FAM19A5. Briefly, binding of anti-FAM19A5 antibodies was assessed by incubating FAM19A5-expressing CHO cells with anti-FAM19A5 antibody at a 1:20 dilution. Cells are washed and binding to PE-labeled anti-human IgG Ab is detected. Flow cytometry analyzes are performed using FACS flow cytometry (Becton Dickinson, San Jose, CA). Preferably, mice that develop the highest titers will be used for fusions.

The ELISA assay as described above can be used to screen for antibodies and thus hybridomas that produce antibodies that exhibit positive reactivity with the FAM19A5 immunogen. Hybridomas that produce antibodies that bind, preferably with high affinity, to FAM19A5 can then be subcloned and further characterized. One clone from each hybridoma that retains parental cell reactivity (by ELISA) can then be selected for cell banking and antibody purification.

To purify antibodies to FAM19A5, selected hybridomas can be grown in two-liter monoclonal antibody purification flasks. Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be tested by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be replaced with PBS and the concentration can be determined from OD 280 using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80°C.

To determine whether selected anti-FAM19A5 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected using a streptavidin-labeled probe. Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using FAM19A5 coated ELISA plates as described above.

To determine the isotype of purified antibodies, an isotype ELISA can be performed using

- 45 043381 reagents specific for antibodies of a certain isotype. For example, to determine the isotype of a human monoclonal antibody, microtiter plate wells can be coated with 1 μg/ml anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plates are reacted with 1 μg/ml or less of the test monoclonal antibodies or purified isotype controls at ambient temperature for 1-2 h. The wells can then be reacted with either human IgG1 or human IgM-specific conjugated with alkaline phosphatase probes. The plates are processed and analyzed as described above.

To test the binding of monoclonal antibodies to live cells expressing FAM19A5, flow cytometry can be used as described in the examples. Briefly, cell lines expressing membrane-bound FAM19A5 (grown under standard growth conditions) were mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 h. After washing, cells were reacted with fluorescein-labeled anti- IgG antibody under the same conditions as the primary antibody staining. Samples can be analyzed by a FACScan instrument using light and side scatter properties to localize individual cells and determine the binding of labeled antibodies. As an alternative analysis using fluorescence microscopy, flow cytometry analysis can be used (in addition to or instead of). Cells can be stained exactly as described above and examined using fluorescence microscopy. This technique allows for the visualization of individual cells, but may have reduced sensitivity depending on antigen density.

Antibodies to FAM19A5 can be further tested for reactivity with the FAM19A5 antigen using Western blotting. Briefly, cell extracts from cells expressing FAM19A5 can be prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum and probed with test monoclonal antibodies. IgG binding can be detected using anti-IgG alkaline phosphatase and processed using BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

Methods for analyzing the binding affinity, cross-reactivity and binding kinetics of various antibodies to FAM19A5 include standard assays known in the art, for example, BIACORE™ surface plasmon resonance (SPR) analysis using the BIACORE™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

In one embodiment, the antibody specifically binds to a soluble form of human FAM19A5. In one embodiment, the antibody specifically binds to the membrane-bound form of human FAM19A5. The antibody may specifically bind to a particular epitope of FAM19A5 (eg, SEQ ID NO: 6 or a fragment within SEQ ID NO: 6). In certain embodiments, the antibody specifically binds human FAM19A5, preferably with high affinity, and does not cross-react with other members of the FAM19 protein subfamily.

IX. Bispecific molecules.

The antibodies described herein can be used to form bispecific molecules. The FAM19A5 antibody or antigen binding portions thereof may be derivatized or linked to another functional molecule, such as another peptide or protein (eg, another antibody or ligand for a receptor) to create a bispecific molecule that binds to at least two different binding sites or molecules -targets. Cytokines such as IL-6, CNTF, LIF, EGF and TGFa have been implicated as triggers for the development of gliosis and/or reactive astrogliosis (Balasingam et al., J. Neurosci. 14(2):846-56 (1994); Winter et al., Proc. Natl. Acad. Sci. U.S.A. 20;92(13):5865-9 (1995)) by activating the protein signal transducer and activator of transcription 3 (STAT3), which then regulates many aspects of reactive astrogliosis after CNS injury . Herrmann J.E. et al., J. Neurosci. 28(28):7231–7243 (2008). For example, absence or reduction of STAT3 results in attenuated up-regulation of glial fibrillary acidic protein (GFAP), failure of astrocyte hypertrophy and increased spread of inflammation, increased lesion volume, and partial attenuation of motor recovery after CNS injury. Herrmann J.E. et al., J. Neurosci. 28(28):7231–7243 (2008). Thus, for example, an anti-FAM19A5 antibody may be coupled to an antibody or scFv that specifically binds to any protein that is involved in inhibiting the development of gliosis and/or overproliferation of reactive astrogliosis for combination treatment, e.g., antibodies to IL-6 CNTF, LIF , EGF or TGFa.

In addition, an anti-FAM19A5 antibody may be associated with an antibody or scFv that treats a disease or disorder, including central nervous system injury (eg, traumatic brain injury, spinal cord injury, stroke, or brain tumor), spinal cord injury, degenerative brain disorder (eg, Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), degenerative cerebrospinal or nerve disorder, or neuropathic pain in the subject (see diseases or disorders in

- 46 043381 section XIII below). For example, an anti-FAM19A5 antibody may be coupled to an antibody or scFv, e.g.

Natalizumab (Tysabri) or Alemtuzumab (Lemtrada), which treats multiple sclerosis.

An antibody described herein may actually be derivatized or linked to more than one other functional molecule to create multispecific molecules that bind to more than two different binding sites and/or target molecules; It is intended that such multispecific molecules are also encompassed by the term bispecific molecule as used herein. To create a bispecific molecule described herein, an antibody described herein can be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent linkage, or otherwise) to one or more other binding molecules, such as another antibody, its an antigen-binding moiety, peptide, or binding mimetic such that a bispecific molecule is obtained. In one embodiment, the bispecific molecule binds to FAM19A5 and VEGF. In another embodiment, the bispecific molecule binds to FAM19A5 and EGF.

Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for FAM19A5 and a second binding specificity for a second target epitope. In an embodiment described herein in which the bispecific molecule is multispecific, the molecule may further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise as binding specificity at least one antibody or antigen binding portion thereof, including, for example, Fab, Fab', F(ab') ₂ , Fv or single chain Fv (ScFv) . The antibody may also be a dimer of a light chain or a heavy chain, or any minimal fragment thereof, such as an Fv or single chain construct as described in Ladner et al. US Pat. No. 4,946,778, the contents of which are expressly incorporated herein by reference.

While human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules described herein are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules described herein can be prepared by conjugating the binding specificities of the components using methods known in the art. For example, each binding specificity of a bispecific molecule can be created separately and then conjugated to each other. When the binding specificities are proteins or peptides, various binding or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-Sacetylthioacetate (SATA), 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3- (2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl-4-(Mmaleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al. (1984), J. Exp. Med. 160:1686 ; Liu, M. A. et al. (1985), Proc. Natl. Acad. Sci. USA, 82:8648). Other methods include those described in Paulus (1985), Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985), Science, 229:81-83) and Glennie et al. (1987), J. Immunol. 139:2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Illinois).

When the binding specificities are antibodies, they can be conjugated through sulfhydryl binding at the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues for conjugation, preferably one. Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is an mAbxmAb, mAbxFab, mAbx(scFv) ₂ , FabxF(ab') ₂ fusion protein or a χFab ligand. The bispecific antibody may comprise an antibody containing an scFv at the C-terminus of each heavy chain. A bispecific molecule described herein may be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may contain at least two single-chain molecules. Methods for producing bispecific molecules are described, for example, in US patent No. 5260203; 5455030; 4881175; 5132405; 5091513; 5476786; 5013653; 5258498 and 5482858.

The binding of bispecific molecules to their specific targets can be confirmed using methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, bioassay (eg, growth inhibition) or Western Blot assay. Each of these assays typically detects the presence of protein-antibody complexes of particular interest using a labeled reagent (eg, antibody) specific for the complex of interest.

- 47 043381

X. Diagnostics.

In one embodiment, the moiety attached to the anti-FAM19A5 antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety. The antibodies described herein can be used for diagnostic purposes, including sample analysis and in vivo imaging, and for this purpose, the antibody (or binding portion thereof) can be conjugated to a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable agents are detectable labels that include radioisotopes for whole body imaging, and radioisotopes, enzymes, fluorescent labels and other suitable antibody labels for testing samples. Detectable labels can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels, including metal sols such as colloidal gold, isotopes such as I ¹²⁵ or Tc ⁹⁹ presented, for example, with a peptide chelator agent N2S2, N3S or N4 type, chromophores, including fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzyme tags that convert the substrate into a detectable marker, and polynucleotide tags that are detected after amplification such as polymerase chain reaction. Suitable enzyme tags include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label may be the enzyme alkaline phosphatase, determined by measuring the presence or production of chemiluminescence following the conversion of 1,2-dioxetane substrates such as adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), disodium-3-(4-methoxyspiro{1,2-dioxetane3,2'- (5'-chloro)tricyclo{3.3.1.1.3.7}decane}-4-yl)phenylphosphate (CSPD), as well as CDP and CDP-STAR® or other luminescent substrates well known to those skilled in the art, such as chelates of suitable lanthanides , such as terbium(III) and europium(III). The detection means is determined by the selected label. The manifestation of the label or its reaction products can be observed with the naked eye when the label is coarse and accumulates at appropriate levels, or by using instruments such as a spectrophotometer, luminometer, fluorimeter, etc., all in accordance with standard practice.

The antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC).

Suitable therapeutic agents include agents that modulate the development of gliosis and/or reactive astrogliosis and/or treat degenerative brain disorders, central nervous system damage, or neuropathic pain. Therapeutics for the treatment of degenerative diseases of the brain include drugs for the treatment of Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis and amyotrophic lateral sclerosis (ALS). This includes drugs commonly used to treat such degenerative brain diseases, such as the drugs described below in section XII.

Immunoconjugates can be prepared by methods known in the art. Preferably, conjugation methods result in the formation of bonds that are substantially (or nearly) non-immunogenic, for example peptide (ie, amide), sulfide, (sterically hindered), disulfide, hydrazone and ester bonds. These bonds are essentially non-immunogenic and exhibit acceptable stability in serum (see, for example, Senter, P.D., Curr. Opin. Chem. Biol. 13 (2009), 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the fragment and antibody, different conjugation strategies can be used. In the case of a naturally occurring fragment or recombinant of 50-500 amino acids, there are standard procedures in textbooks describing the technology for the synthesis of protein conjugates, which can be easily performed by a person skilled in the art (see, for example, Hackenberger, C.P.R. and Schwarzer D., Angew Chem Int Ed Engl 47 (2008), 10030-10074). In one embodiment, the reaction of a maleimide moiety with a cysteine residue in the antibody or fragment is used. This is a particularly suitable coupling technology if, for example, a Fab or Fab' fragment of an antibody is used. Alternatively, in one embodiment, the connection is made to the C-terminal end of the antibody or fragment. C-terminal modification of a protein, such as a Fab fragment, for example, can be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009), 3361-3371).

Typically, site-specific reaction and covalent binding are based on the conversion of a naturally occurring amino acid into an amino acid with a reactivity that is independent of the reactivity of other functional groups present. For example, a particular cysteine in the context of a rare sequence can be enzymatically converted to an aldehyde (see Frese, M.A., and Dierks, T., ChemBioChem. 10 (2009), 425-427). It is also possible to obtain the desired amino acid modification by exploiting the specific enzymatic reactivity of certain enzymes with the natural amino acid in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004), 119-126 ; Gautier, A. et al., Chem. Biol. 15 (2008), 128-136; and protease-catalyzed C-N bond formation used in Bordusa F., Highlights in Bioorganic Chemistry (2004), 389-403 ).

Site-specific reaction and covalent binding can also be achieved by selectively reacting terminal amino acids with appropriate modifying reagents. The reactivity of N-terminal cysteine with benzonitriles (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009), 9658-9662) can be used to achieve site-specific covalent binding. Native chemical ligation can also rely on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

EP 014453 describes a conjugation method which is based on a faster reaction of a cysteine within a region of negatively charged amino acids with a cysteine located in a region of positively charged amino acids.

The fragment may also be a synthetic peptide or a peptide mimic. If the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity may be incorporated during such synthesis (see, for example, de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009), 1281-1295). Since a wide variety of orthogonal functional groups are involved and can be incorporated into a synthetic peptide, conjugation of such a peptide to a linker is a standard technique.

To obtain a monolabeled polypeptide, the 1:1 stoichiometry conjugate can be separated by chromatography from other conjugation byproducts. This procedure can be facilitated by using a dye-labeled member of the binding pair and a charged linker. By using this type of labeled and highly negatively charged member of a binding pair, monoconjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides that carry more than one linker, since the difference in charge and molecular weight can be used for separation. A fluorescent dye may be useful in purifying the complex from unbound components, such as the labeled monovalent binder.

XI. Pharmaceutical compositions.

Provided herein are compositions containing the antibody or antigen binding portion thereof described herein, having the desired degree of purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes) and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In a specific embodiment, the pharmaceutical compositions contain an antibody or antigen-binding portion thereof, a bispecific molecule or immunoconjugate described herein, and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical compositions contain an effective amount of an antibody or antigen binding portion thereof described herein, and optionally one or more additional prophylactic therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for enhancing, inducing or activating FAM19A5 activity and treating a condition such as central nervous system damage, degenerative brain disorder or neuropathic pain.

Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable agents. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include vegetable fatty oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multi-dose containers, which include phenols or cresols, mercury compounds, benzyl alcohol, chlorobutanol esters, methyl and propyl p-hydroxybenzoic acid, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifiers include Polysorbate 80 (TWEEN® 80). The metal ion sequestering or chelating agent includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible carriers, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenteral, intrathecal, intracerebroventricular, pulmonary, subcutaneous, or intraventricular. Parenteral administration, characterized by either subcutaneous, intramuscular, or intravenous injection, is also discussed herein. Injectable preparations may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions for administration may also contain minor amounts of non-toxic excipients such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers and other agents such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Formulations for parenteral administration of the antibody include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a diluent immediately before use, including tablets for subcutaneous injection, sterile suspensions, ready for injection, sterile dry insoluble products, ready for use together with the carrier immediately before use and sterile emulsions. Solutions may be aqueous or non-aqueous.

For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

Topical mixtures containing the antibody are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion or the like. and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, drenches, sprays, suppositories, dressings, skin patches, or any other formulations suitable for topical applications. The antibody or antigen-binding portion thereof described herein can be formulated as an aerosol for topical administration, for example, by inhalation (see, for example, US Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which disclose aerosols for delivering a steroid that is used for the treatment of inflammatory diseases, in particular asthma). These respiratory compositions may be in the form of an aerosol or nebulizer solution or as a fine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the drug particles in one embodiment have diameters less than 50 microns, in one embodiment less than 10 microns.

The antibody or antigen-binding portion thereof described herein may be formulated for topical or topical use, such as topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams and lotions, and for ocular or intracisternal application. or intraspinal use. Topical administration is envisaged for transdermal delivery, as well as for ocular or mucosal administration or for inhalation therapy. Nasal antibody solutions may also be administered alone or in combination with other pharmaceutically acceptable excipients.

Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art and can be used to administer the antibody. For example, such patches are described in US patents No. 6267983, 6261595, 6256533, 6167301, 6024975, 6010715, 5985317, 5983134, 5948433 and 5860957.

In certain embodiments, the pharmaceutical composition containing the antibody or antigen-binding moiety described herein is a lyophilized powder that can be reconstituted for administration as solutions, emulsions, and other mixtures. It can also be reconstituted and formed into solids or gels. The lyophilized powder is prepared by dissolving the antibody or antigen binding portion thereof described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. IN

- 50 043381 In some embodiments, the lyophilized powder is sterile. The solvent may contain a filler that improves stability or other pharmacological component of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment, at approximately neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art yields the desired composition. In one embodiment, the resulting solution will be distributed into vials for lyophilization. Each vial will contain one dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, for example at a temperature of from about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral use. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the selected compound. This amount can be determined experimentally.

The antibodies or antigen-binding portions thereof, bispecific molecule or immunoconjugate described herein, and other compositions provided herein may also be formulated to target a specific tissue, receptor, or other area of the body of the subject being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in instant formulations. For non-limiting examples of targeting methods, see, for example, US Patent Nos. 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751, 6071495, 6060082, 6048736, 6039975,60 04534,5985307,5972366, 5900252, 5840674, 5759542 and 5709874. In a specific embodiment, the antibody or antigen-binding portion thereof described herein is aimed at treating central nervous system injury, degenerative brain disease, or neuropathic pain.

Compositions used for in vivo administration may be sterile. This is easily achieved by filtration, for example through sterile filter membranes.

XII. Sets.

Provided herein are kits containing one or more of the antibodies described herein, or antigen binding portions thereof, bispecific molecules, or immunoconjugates thereof. In a specific embodiment, provided herein is a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, such as one or more antibodies provided herein, or an antigen binding portion thereof, optionally instructions for use. In some embodiments, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent such as those described herein.

XIII. Therapeutic uses and methods.

In one aspect, provided herein are methods of mitigating injury or damage to the CNS in a subject, comprising administering to a subject in need thereof an antibody to FAM19A5 or an antigen-binding portion thereof, a bispecific molecule or immunoconjugate described herein, or a composition thereof.

In one embodiment, provided herein are methods for inhibiting, delaying, suppressing, limiting, reducing, eliminating, or preventing the onset or initiation of gliosis and associated detrimental effects on the CNS in a subject, comprising administering to the subject an antibody to FAM19A5 or an antigen-binding portion thereof, a bispecific molecules or immunoconjugates described herein, or compositions thereof. In one embodiment, provided herein are methods for inhibiting, slowing, suppressing, limiting, reducing, eliminating, or preventing excessive or abnormal proliferation of reactive astrocytes and associated detrimental effects on the CNS in a subject, comprising administering to the subject an anti-FAM19A5 antibody or an antigen-binding portion thereof , a bispecific molecule or immunoconjugate described herein, or a composition thereof. In one embodiment, provided herein are methods for reducing, inhibiting, or reducing the expression of chondroitin sulfate proteoglycans (including the level of Neurocan, NG2, or both) or reducing the activity or causing inactivation of Neurocan, NG2, or both in a subject, comprising administering to the subject an antibody to FAM19A5 or an antigen-binding portion thereof, a bispecific molecule or immunoconjugate described herein, or a composition thereof. In one embodiment, provided herein are methods for stimulating, promoting, increasing or activating neuronal growth, preferably following injury or damage in a subject, comprising administering to the subject an antibody to FAM19A5 or an antigen binding portion thereof, a bispecific molecule

- 51 043381 ly or immunoconjugates described herein, or compositions thereof. In one embodiment, provided herein are methods for increasing the level of c-fos mRNA, c-fos protein or c-fos protein activity and increasing the level of ERK mRNA, ERK protein or pERK activity, preferably in neuronal nuclei, in a subject in need thereof comprising administering to a subject an antibody to FAM19A5 or an antigen-binding portion thereof, a bispecific molecule or immunoconjugate described herein, or a composition thereof. In one embodiment, provided herein are methods for increasing or increasing the level of GAP43 mRNA, GAP43 protein, or increasing GAP43 protein activity, preferably in neurons in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody or an antigen-binding portion thereof, a bispecific molecule, or immunoconjugate described herein, or a composition thereof. In one embodiment, provided herein are methods for enhancing or promoting neuronal survival and/or promoting axon outgrowth in a subject in need thereof, comprising administering to the subject an anti-FAM19A5 antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein, or their compositions. In one embodiment, the subject is a human, preferably a human with neuronal injury or damage, such as from CNS injury, trauma, lesion, spinal cord injury, brain tumor, infection, ischemia, stroke, autoimmune responses and/or neurodegenerative diseases.

In some embodiments, provided herein are methods for treating a disease or disorder, including a central nervous system injury, a spinal cord injury, a degenerative brain disorder, a degenerative cerebrospinal or neural disorder, or neuropathic pain in a subject in need thereof, comprising administering to the subject an antibody to FAM19A5 or an antigen binding portion, bispecific molecule or immunoconjugate described herein, or a composition thereof. In one embodiment, the central nervous system injury is traumatic brain injury, spinal cord injury, stroke, brain tumor (eg, glioma or glioblastoma), or a combination thereof. In one embodiment, the degenerative brain disorder is Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), or combinations thereof. Thus, in one embodiment, described herein is a method for treating traumatic brain injury, spinal cord injury, stroke, brain tumor (e.g., glioma or glioblastoma), or a combination thereof in a subject in need thereof, comprising administering to the subject an antibody to FAM19A5 or an antigen binding portion, bispecific molecule or immunoconjugate described herein, or a composition thereof.

In one embodiment, described herein is a method for treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS in a subject in need thereof, comprising administering to the subject an antibody to FAM19A5 or an antigen binding portion thereof, a bispecific molecule, or an immunoconjugate described herein document, or their composition. In some embodiments, the subject is a human.

In some embodiments, the methods described herein can be used to treat a brain tumor, where the methods include administering an anti-FAM19A5 antibody (eg, one described herein) to a subject in need thereof. In certain embodiments, the brain tumor is a glioma or glioblastoma.

In some embodiments, administration of an anti-FAM19A5 antibody reduces tumor growth (eg, glioblastoma) compared to a control condition (eg, the corresponding value in a subject who did not receive the anti-FAM19A5 antibody). In certain embodiments, tumor growth (e.g., tumor volume or weight) is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90 or 100% compared to the control condition (e.g., the corresponding value in a subject who did not receive antibody to FAM19A5).

In some embodiments, administration of an anti-FAM19A5 antibody helps normalize blood vessels in a tumor (eg, glioblastoma). In certain embodiments, normalization of blood vessels includes (i) increased number of blood vessels, (ii) improved blood vessel communication, (iii) decreased blood vessel permeability, (iv) increased blood flow velocity, or (v) any combination thereof. In certain embodiments, the methods described herein increase the number of blood vessels (with increased thickness and improved communication) that extend into the tumor by at least 5, 10, 20, 30, 40, 50, 60, 70, 80 , 90 or 100% or more.

In some embodiments, administration of an anti-FAM19A5 antibody increases immune cell infiltration into a tumor (eg, glioblastoma). In some embodiments, immune cell infiltration into the tumor is increased by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more compared to the control condition (e.g., the number of immune cells

- 52 043381 present in a tumor that was not exposed to anti-FAM19A5 antibody). In some embodiments, immune cells include macrophages, dendritic cells, microglia, and T lymphocytes. In additional embodiments, the methods described herein increase the cellular volume of immune cells present in a tumor. In some embodiments, the cellular volume of immune cells is increased by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more compared to the control condition (e.g., the volume of immune cells present in a tumor that was not exposed to anti-FAM19A5 antibody).

In some embodiments, administration of an anti-FAM19A5 antibody (eg, those described herein) increases survival of a subject with a tumor (eg, a brain tumor). In some embodiments, survival is increased by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more compared to a control condition (e.g., the corresponding value in a subject who did not receive antibody to FAM19A5). In some embodiments, survival is increased by at least 1, 2, 3, 4, 5, 6, 7 days, 2, 3, 4 weeks, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12 months, 2, 3, 4 years or 5 years or more.

In some embodiments, the brain tumor (eg, glioblastoma) that can be treated using the present methods may be metastatic, inoperable, and refractory to prior cancer therapy. In certain embodiments, prior cancer therapy includes chemotherapy. In some embodiments, the chemotherapy includes platinum-based therapy. In some embodiments, the platinum-based therapy includes a platinum-based anticancer drug selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. In certain embodiments, the platinum-based therapy includes cisplatin. In additional embodiments, the platinum-based therapy includes carboplatin. In some embodiments, prior cancer therapy includes immunotherapy (eg, anti-PD-1 antibody).

In some embodiments, anti-FAM19A5 antibodies useful in the methods of the invention (eg, for treating a brain tumor, eg, glioblastoma) may be administered in combination with other compounds, drugs, and/or agents used to treat cancer. Such compounds, drugs and/or agents may include, for example, chemotherapeutic drugs, small molecule drugs or antibodies that stimulate an immune response to the cancer. In some embodiments, the methods described herein are used in combination with standard treatment (eg, surgery, radiation, and chemotherapy). In other embodiments, the methods described herein are used as maintenance therapy, such as therapy intended to prevent the occurrence or recurrence of tumors.

In some embodiments, the anti-FAM19A5 antibodies used for the present invention may be combined with more than one immuno-oncology agent and may, for example, be combined with a combinatorial approach that targets multiple elements of the immune pathway, such as one or more of the following: therapy that enhances tumor antigen presentation (eg, dendritic cell vaccine, GMCSF-secreting cell vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit immune downregulation, for example, by inhibiting the CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or other immunosuppressive cells (eg, suppressor myeloid cells); therapies that stimulate positive immune regulation, for example, using agonists that stimulate the CD-137, OX-40 and/or CD40 or GITR pathway and/or stimulate T cell effector function; therapies that systemically increase the frequency of antitumor T cells; therapy that depletes or inhibits Tregs, such as Tregs in a tumor, for example, using a CD25 antagonist (eg, daclizumab) or by depleting anti-CD25 beads ex vivo; therapies that affect the function of myeloid suppressor cells in the tumor; therapy that enhances the immunogenicity of tumor cells (eg, anthracyclines); transfer of adoptive T cells or NK cells, including genetically modified cells, such as chimeric antigen receptor modified cells (CAR-T therapy); therapy that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; therapies that reverse/prevent T cell energy or exhaustion; therapies that induce innate immune activation and/or inflammation at the tumor site; administering immunostimulatory cytokines or blocking immunorepressive cytokines. In some cases, an additional anti-cancer agent that can be combined with the anti-FAM19A5 antibodies used herein may include, but is not limited to, one or more anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-OX40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L), anti-CD137, anti-LAG-3, anti-GITR, or any combination thereof.

In certain embodiments, the anti-FAM19A5 antibodies described herein are

- 53 043381 can treat, reduce, eliminate, prevent, attenuate, control or inhibit fibrosis in a subject in need thereof, including antibodies to FAM19A5 or an antigen-binding portion thereof. In one embodiment, the fibrosis is benign (ie, does not cause symptoms). In another embodiment, fibrosis is associated with a pathological condition that may or may not be reversible. In some embodiments, the fibrosis is selected from the group consisting of liver fibrosis, pulmonary fibrosis, renal fibrosis, myelofibrosis, pancreatic fibrosis, skin fibrosis, myocardial fibrosis, arterial fibrosis, arthrofibrosis, mammary fibrosis, muscular fibrosis, retroperitoneal fibrosis, thyroid fibrosis , lymph node fibrosis, bladder fibrosis and pleural fibrosis. In one embodiment, the fibrosis is liver fibrosis or pulmonary fibrosis. In another embodiment, fibrosis is associated with a tumor caused by cancer, such as liver cancer, lung cancer, kidney cancer, breast cancer, brain cancer and/or pancreatic cancer. In one embodiment, the method reduces, eliminates, alleviates, improves, inhibits, or slows or prevents fibrosis, a symptom associated with fibrosis, an underlying cause of fibrosis, or a combination thereof.

In other embodiments, anti-FAM19A5 antibodies described herein can treat, reduce, eliminate, prevent, attenuate, control, or inhibit a fibrosis-related disease or disorder in a subject in need thereof, including anti-FAM19A5 antibodies or an antigen-binding portion thereof .

The term fibrosis associated with a disease or disorder refers to fibrosis that accompanies a disease or disorder or is caused by or resulting from a disease or disorder. The term includes fibrosis arising from or caused by a disease or disorder described above, or fibrosis accompanying a disease or disorder.

In yet other embodiments, the anti-FAM19A5 antibodies described herein can treat, reduce, eliminate, prevent, delay, improve, control, or inhibit liver cancer, lung cancer, kidney cancer, breast cancer, brain cancer (eg, glioma or glioblastoma) and/or pancreatic cancer in a subject in need thereof, including administering to the subject an antibody to FAM19A5 or an antigen-binding portion thereof. In some embodiments, liver cancer, lung cancer, kidney cancer, breast cancer, brain cancer, and/or pancreatic cancer are associated with or caused by fibrosis. In some embodiments, the brain tumor is a glioma or glioblastoma.

In some embodiments, a therapeutically effective amount of an anti-FAM19A5 antibody or antigen-binding moiety, bispecific molecule, or immunoconjugate described herein is administered. When treating a subject (eg, a human), the therapeutically effective amount of an anti-FAM19A5 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein depends on factors such as age, sex, and extent of disease. Typically, the anti-FAM19A5 antibody or antigen-binding portion, bispecific molecule, or immunoconjugate described herein is administered at a dose of between 0.01 μg and 1000 mg per day.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding moiety, bispecific molecule or immunoconjugate, or composition thereof described herein is administered intravenously, orally, parenterally, transthecally, intrathecally, intracerebroventricularly, pulmonaryly, subcutaneously, or intraventricularly.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion or composition thereof may be administered in combination with one or more additional agents for the treatment of fibrosis (eg, pirfenidone (ESBRIET®) or nintedanib (OFEV®) for idiopathic pulmonary fibrosis). The dosage and administration of one or more additional therapeutic drugs are known in the art, for example, as indicated on the product label of the corresponding drug.

In some embodiments, an anti-FAM19A5 antibody or antigen-binding portion thereof, or a composition thereof, may be administered in combination with one or more additional agents to treat a central nervous system injury (e.g., traumatic brain injury, spinal cord injury, stroke, or brain tumor (e.g., glioma or glioblastoma)), spinal cord injury, degenerative brain disorder (eg, Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), degenerative cerebrospinal or nerve disorder, or neuropathic pain. For example, non-limiting typical agents for the treatment of Huntington's disease include tetrabenazine (XENAZINE®), antipsychotic drugs such as haloperidol (HALDOL®), chlorpromazine, risperidone (RISPERDAL®) and quetiapine (SEROQUEL®).

Non-limiting typical agents for the treatment of Parkinson's disease include levodopa (with or without carbidopa), dopamine agonists such as pramipexole (MIRAPEX®), ropinirole (REQUIP®), and rotigotine (NEUPRO®), and apomorphine (APOKYN®), selegiline ELDEPRYL® and ZELAPAR®), rasagiline

- 54 043381 (AZILECT®), entacapone (COMTAN®), benztropine (COGENTIN®), trihexyphenidyl and amantadine.

Non-limiting exemplary agents for the treatment of Alzheimer's disease include donepezil (ARICEPT®), galantamine (RAZADYNE®), and rivastigmine (EXELON®).

Non-limiting typical agents for the treatment of multiple sclerosis include glatiramer acetate (COPAXONE®), dimethyl fumarate (TECFIDERA®), fingolimod (GILENYA®), teriflunomide (AUBAGIO®), natalizumab (TYSABRI®), alemtuzumab (LEMTREMA®), and mitoxantrone.

Non-limiting typical agents for the treatment of ALS include riluzole (RILUTEK®).

The dosage and administration of one or more additional therapeutic drugs are known in the art, for example, as indicated on the product label of the corresponding drug.

The following examples are offered by way of illustration and not by way of limitation.

Examples

Experimental methods and details are given in the following examples.

Example 1: Expression and purification of human FAM19A5 protein.

Recombinant human FAM19A5 protein was produced and purified, and the purified protein was used in an antibody screening assay based on binding affinity analysis. First, the LPS-hT plasmid containing the FAM19A5 gene was transformed into bacteria and induced to overexpress the protein. Once produced, the FAM19A5 protein was purified using Ni-NTA affinity chromatography (Qiagen, Valencia, CA, USA). Using progressively higher concentrations of imidazole, the His-tagged FAM19A5 protein was removed from the Ni column. Protein expression in solution was measured using Coomassie Brilliant Blue R-250 dye. Taking only the solution containing FAM19A5 imidazole, the FAM19A5 protein was concentrated using PBS. Once the concentration was adjusted, the purity and concentration of FAM19A5 protein was measured using Western blot analysis. The concentrated protein was then used to screen for antibodies specific to FAM19A5.

Example 2. Preparation of FAM19A5 antibody libraries.

Cell wall components TDW and CWS containing a water-in-oil emulsion adjuvant (RIBI+MPL+TDM+ CWS adjuvant, Sigma, St. Louis, MO, USA) were emulsified and then subcutaneously injected into three chickens. The chickens were immunized a total of three times with an interval of approximately 2-3 weeks between immunizations. Antibody titers obtained from immunized chickens were measured by immunoblotting using lysates of HEK293T cells that overexpressed the FAM19A5 protein.

A single-chain variable fragment (scFv) library was prepared from immunized chicken using TRI reagent (Invitrogen, Carlsbad, CA, USA), and RNA was extracted from the spleen, bone marrow, and synovium of the immunized chickens described above. Oligo-dT primers and the SUPERSCRIPT™ III First-Strand Synthesis System (Invitrogen) were used to synthesize first-strand cDNA. For cDNA obtained from the chicken immune system, the Expand High Fidelity PCR System (Roche Molecular Systems, Indiana, USA) was used to generate a library of single chain variable regions. In each reaction, 1 μl of cDNA, 60 pmol of each primer, 10 μl of 10× reaction buffer, 8 μl of 2.5 mM dNTPs (Promega, Madison, WI, USA) and 0.5 μl of Taq DNA polymerase were mixed with water. The final volume was 100 μl. The PCR reaction was carried out using the following conditions: 30 cycles: (i) 15 s at 94°C, (ii) 30 s at 56°C and (iii) 90 s at 72°C followed by a final extension of 10 min at 72 °C. PCR products containing an approximately 350-bp fragment were loaded onto a 1.5% agarose gel and, after electrophoresis, a QIAGEN Gel II Extraction Kit (QIAGEN, Valencia, CA, USA) was used to purify the nucleotide fragment. The purified PCR product was quantified by reading at OD 260 nm (1 OD unit = 50 μg/mL).

The first two products VH and VL from the second PCR were randomly linked using Overlap extension PCR. Each PCR reaction was mixed with 100 ng of purified VL and VH products, 60 pmol of each primer, 10 μl of 10x reaction buffer, 8 μl of 2.5 mM dNTPs, 0.5 μl of Taq DNA polymerase, and water in a final volume of 100 μl. PCR was performed using the following conditions: 25 cycles: (i) 15 s at 94°C, (ii) 30 s at 56°C, and (iii) 2 min at 72°C, followed by a final extension of 10 min at 72°C WITH. PCR products containing an approximately 700-bp single-stranded variable region fragment were loaded onto a 1.5% agarose gel and, after electrophoresis, a QIAGEN Gel II Extraction Kit (QIAGEN) was used to purify the nucleotide fragment. The purified PCR product was quantified by reading at OD 260 nm (1 OD unit = 50 μg/mL).

The scFv fragment of the PCR product and the pComb3X-SS vector (The Scripps Research Institute, California, USA) were digested with Sfi restriction enzyme. 10 μg of purified overlap PCR product was mixed with 360 units of Sif I (1 μg DNA per 16 units, Roche Molecular Systems, Pleasanton, CA, USA), 20 μl of 10x reaction buffer, and water to a final volume of 200 μl. 20 μg of pComb3X-SS vector was mixed with 120 units of Sfi I (1 μg of DNA per 6 units), 20 μl of 10x reaction buffer

- 55 043381 and water to a final volume of 200 µl. The mixture was digested at 50°C for 8 hours. The digest containing the scFv fragment (approximately 700 bp) and vector (approximately 3400 bp) was then loaded onto a 1% agarose gel and purified using QIAGEN Gel II Extraction Kit (QIAGEN, Valencia, CA, USA). 1400 ng Sfi-restricted vector pComb3X and 700 ng digested scFv fragments were mixed with 5× ligation buffer, 10 μl T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) and water to a final volume of 200 μl. The mixture was incubated at 16°C for 16 hours to perform ligation.

After ethanol precipitation, the DNA precipitate was dissolved in 15 μL of water. To obtain the library, the ligation sample was transformed into E. coli strain ER2738 (New England Biolabs Inc, Hitchin, Hertfordshire, SG4 OTY, England, UK) by electroporation using a gene pulser (Gene pulser: Bio-Rad Laboratories, Hercules, CA, USA ). Cells were mixed in 5 ml of Super Broth (SB) medium and incubated with agitation at 250 rpm for 1 h at 37°C. Then, 3 μl of 100 mg/ml kanamycin was added to 10 ml of SB medium. To determine library size, 0.1, 1, and 10 μL of culture sample were spread onto Luria Broth (LB) agar plates containing 50 μg/mL kanamycin. After stirring for 1 hour, 4.5 μl of 100 mg/ml kanamycin was added to the LB culture and stirred for an additional 1 hour. Then 2 ml of helper phage VCM13 in water (>1011 CFU/ml) was added to the LB medium along with prewarmed LB (183 ml) containing 92.5 μl of 100 mg/ml kanamycin. This mixture was stirred at 250 rpm at 37°C for an additional 2 hours. Then 280 μl (50 mg/ml) of kanamycin was added to the culture and stirred overnight at 37°C. The next day, the bacterial sediment was centrifuged using a high-speed centrifuge (Beckman, JA-10 rotor) at 3000g, 4°C. The bacterial pellet was then used to extract phagemid DNA, and the supernatant was transferred into sterile vials for centrifugation. Then, 8 g of polyethylene glycol-8000 (PEG-8000, Sigma) and 6 g of sodium chloride (NaCl, Merck) were added to the supernatant and then kept for 30 min on ice. After this, the supernatant was centrifuged for 15 min at 15000g, 4°C. The supernatant was then discarded and the phage pellet with Tris containing 1% BSA replication was suspended in buffered saline (TBS).

Example 3. Panning library (biopanning) on immobilized antigen.

Biopanning was performed using magnetic beads (Dynabeads M - 270 Erochu, Invitrogen). At room temperature, approximately 1 x 10 ⁷ beads were coated with 5 μg of recombinant FAM19A5 protein by rotating the beads and protein together for 20 hours at room temperature. After coating, the beads were washed 4 times with phosphate-buffered saline (PBS) and blocked for 1 h in PBS containing 3% BSA at room temperature. The coated beads were then cultured for 2 h at room temperature with scFv after phage display as described above. To remove any phage that was not bound to the antigen-coated beads, the beads were washed with 0.05% Tween20/PBS. Bound phages were then eluted with 50 µl 0.1 M glycine/hydrogen chloride (0.1 M glycine-HCl, pH 2.2) and neutralized with 3 µl 2 M Tris-hydrogen chloride (Tris-HCl, pH 9.1) . These phage-containing supernatants were used to infect E. coli ER2738 cells, and helper phage VCSM13 was used for overnight amplification and recovery. In addition, input (input) and production (output) of phage titers from phage-infected cultures were determined by blotting phage-infected cultures onto LB agar plates containing 50 μg/ml kanamycin. The next day, PEG-8000 and NaCl were used to precipitate the phages that were subsequently used for biopanning. Biopanning was performed up to five times in total, repeating the above process. For each amplification, phages were screened and selected for high affinity for the FAM19A5 protein.

Example 4: Clone selection using phage ELISA.

To analyze the clones selected from the biopanning, individual clones were randomly selected from scFv after phage display and confirmed by ELISA that the clones bound the recombinant FAM19A5 protein. Recombinant FAM19A5 protein was diluted in 0.1 M NaHCO ₃ buffer and 100 ng/well of protein was used to coat 96-well microtiter plates at 4°C for 16 hours. The next day, the plates were blocked with 3% BSA/PBS at 37°C for 1 hour. The phage supernatant was then mixed with 6% BSA/PBS and cultured for 2 hours at 37°C. The plates containing the supernatant were then washed with 0.05% Tween20/PBS. HRP-conjugated antibody M13 (a-M13-HRP, Pierce Chemical Co, Rockford, IL, USA) was diluted to 1/5000. 50 μl of diluted antibody was added to the plates and incubated for 1 h at 37°C. After incubation and washing, 0.05 M citrate buffer solution, 1 μg/ml 2,2'-azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS, Amresco, Solon, OH, USA) and 0.05 M citrate buffer were added to the plates. 1% H ₂ O ₂ for color formation. The absorbance for each well was measured at 405 nm.

Then, 24 clones that bind to the recombinant FAM19A5 protein and exhibit high absorption were analyzed, and from these, 13 scFv clones with unique sequences were obtained. After further selection, clones 2-13, 1-28 and 3-2 were obtained.

Example 5. Preparation of antibody to FAM19A5-IgG2/4.

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Ahtu-FAM19A5 scFv was subcloned into a mammalian expression vector. In the scFv gene sequence FAM19A5, the human Sk gene was linked to the light chain variable domain, and the human immunoglobulin isotype IgG2/4 genes CH1, CH2 and CH3 were linked to the heavy chain variable region. An antibody having each light chain and each heavy chain was synthesized by adding restriction sites (Genscript, USA). The synthesized gene was inserted into a mammalian cell expression vector containing a modified restriction site to facilitate cloning. First, the light chain gene was inserted into the vector using the restriction enzymes Hind III and Xba I (New England Biolabs, UK), and then the heavy chain gene was added to the vector using the restriction enzymes NheI and BamHI (New England Biolabs, UK).

An injection system for transfection and overexpression in mammalian cells was used to express and purify the anti-FAM19A5-IgG2/4 antibody. Mix 2 μg/ml mammalian expression vector with 4 μg polyethylenimine (PEI, Polysciences, Warrington, PA, USA) in 150 mM sodium chloride (NaCl, Merck), corresponding to 1/10 the volume of the cell culture. The mixture was left for 15 minutes at room temperature. The mixture was added to HEK293F cells (2 x 10 ⁶ cells/ml, Invitrogen), which were then incubated in FREESTYLE™ 293 expression culture medium containing IOOU/ml penicillin and streptomycin (Invitrogen) at 7% CO2 and 37°C with 135 rpm agitation. /min for six days. Protein A bead affinity gel chromatography (RepliGen, Waltham, MA, USA) was used to purify expressed anti-FAM19A5 IgG2/4 antibodies from cell culture supernatant. Protein A chromatography was performed using gel electrophoresis with a Bis-Tris gradient of 4-12%. Protein size and yield were confirmed by Coomassie brilliant blue staining.

Example 6: Epitope Mapping Analysis Using FAM19A5 F1-F6 Epitope Fragments.

Overlapping peptide fragments (F1-F6, see Fig. 1) of the human FAM19A5 protein were synthesized and conjugated to BSA. The binding of various anti-FAM19A5 antibodies to BSA-conjugated peptide fragments F1-F6 was determined by ELISA analysis. Briefly, the FAM19A5 F1-F6 fragment (diluted to 1 μg/ml in 50 mM carbonate buffer (Biosesang) or to 20 μg/ml for high concentration assays) was used to coat the wells of 96-well immunoplates (Thermo Scientific) (100 μl/ well) overnight at 4°C and then washed twice in 1x PBS. The plates were then blocked with blocking buffer (100 μl/well) for 1 h at room temperature. During the 1-h incubation, the corresponding anti-FAM19A5 antibodies were diluted to 1 μg/ml (or 20 μg/ml for the high concentration assay) in diluent buffer. After the plates were washed (2x using 1x PBS), diluted anti-FAM19A5 antibodies were added to the appropriate wells and the plates were incubated at room temperature for 1 hour. The plates were then washed a total of five times using wash buffer. The ODP substrate (prepared by dissolving one tablet of ODP (O-phenylenediamine dihydrochloride, Thermo) in 9 ml of sterilized deionized water and 1 ml of 10x stable peroxide stable buffer (Theromo)) was then added to each of the wells and the color change reactions were allowed to occur within 10 min. This reaction was stopped by adding 100 μl of 2N. H ₂ SO ₄ (Daejung) into the wells. The absorbance value of each well was determined at 492 nm using a 96-well microplate reader (Molecular Device).

As shown in FIG. 2, anti-FAM19A5 antibody 3-2 bound strongly to the F2 epitope fragment with minimal binding to other fragments. This was also true for anti-FAM19A5 antibody 1-28 (data not shown). In contrast, antibody 1-65 binds strongly only to the F5 epitope fragment. Interestingly, anti-FAM19A5 antibody 2-13 did not appear to bind to any of the F1-F6 epitope fragments.

Next, to identify the specific amino acid residues in the F2 epitope fragment to which antibodies 3-2 and 1-28 bind, various amino acid residues of the F2 fragment were replaced by alanine or valine, as shown in Table 1. 8. Mutated residues are shown in bold and underlined. The binding affinity of the indicated antibodies to FAM19A5 was measured using an ELISA assay as described above.

Table 8

Mutant peptide (#) Sequences Wild type TLDRDSSQPRRTIARQTARC (SEQ ID NO: 6) F2-01-BSA (#1) TADRDSSQPRRTIARQTARC (SEQ ID NO: 73) F2-02-BSA (#2) TLARDSSQPRRTIARQTARC (SEQ ID NO: 74) F2-03-BSA (#3) TLDRASSQPRRTIARQTARC (SEQ ID NO: 75) F2-04-BSA (#4) TLDRDASQPRRTIARQTARC (SEQ ID NO: 76) F2-05-BSA (#5) TLDRDSAQPRRTIARQTARC (SEQ ID NO: 77)

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F2-06-BSA (#6) TLDRDSSAPRRTIARQTARC (SEQ ID NO: 78) F2-07-BSA (#7) TLDRDSSQARRTIARQTARC (SEQ ID NO: 79) F2-08-BSA (#8) TLDRDSSQPRATIARQTARC (SEQ ID NO: 80) F2-09-BSA (#9) TLDRDSSQPRRAIARQTARC (SEQ ID NO: 81) F2-10-BSA (#10) TLDRDSSQPRRTAARQTARC (SEQ ID NO: 82) F2-11-BSA (#11) TLDRDSSQPRRTIRRQTARC (SEQ ID NO: 83) F2-12-BSA (#12) TLDRDSSQPRRTIAAQTARC (SEQ ID NO: 84) F2-13-BSA (#13) TLDRDSSQPRRTIARQTVRC (SEQ ID NO: 85)

As shown in FIG. 3A and 3B, antibody 3-2 binds strongly to mutant F2 peptide fragments #1,2, 4-6 and 9-13 with reduced binding to fragments #3, 7 and 8. These last three mutant F2 peptide fragments contain an alanine substitution in amino acid residues D5, P9 and R11 (numbered according to SEQ ID NO: 6 in Table 8), respectively, suggesting that antibody 3-2 binds to FAM19A5 primarily at these amino acid residues.

As for antibody 1-28, it bound strongly to mutant F2 peptide fragments #1, 2, 6 and 8-13, whereas with peptide fragments #3, 4, 5 and 7, antibody 1-28 showed a significant decrease in binding. See fig. 3C. These data indicate that the antibody to FAM19A5 1-28 binds to FAM19A5 predominantly at amino acid residues D5, S6, S7 and P9 (numbered according to SEQ ID NO: 6 in Table 8) within the F2 epitope fragment.

Example 7 Epitope mapping analysis using FAM19A5M1-M8 mutants.

To further characterize the binding epitopes of the anti-FAM19A5 antibodies described herein, the amino acid sequences for the various FAM19 family members (ie, FAM19A1-5) were aligned as shown in FIG. 4. Based on this alignment, eight regions were identified where the amino acid sequences of the FAM19A5 protein were most significantly different from other members of the FAM19A family (ie, FAM19A1-4) (M1-M8). The amino acid sequences of these regions were replaced by the consensus sequence of the corresponding regions for the FAM19A1-4 proteins. See table. 8 (mutated amino acid residues are highlighted in bold and underlined).

Mutant FAM19A5-expressing phages were prepared as follows. To prepare the phage culture medium, 55.6 ml of 2 M glucose (D-(+)-glucose, Sigma), 5 ml of 1 M MgCl ₂ (magnesium chloride, Junsei), 1 ml of 34 mg/ml chloramphenicol (Sigma) were added to the medium 2xYT. Colonies obtained by monophage ELISA were selected and placed in 5 ml of prepared medium (2xYT-GMC) for cultivation for 16 h at 37°C in a shaking incubator (VS-8480, Vision). 100 μl of each culture was transferred into 10 ml of 2xYT-GMC separately, which were cultured at 37°C in an incubator until the detection value at OD 600 nm reached 0.5. Once the detection value reached 0.5 at OD 600 nm, 5 mL of each culture was obtained as infection samples. After sample preparation, 50 μl of M1 helper phage was added to individual samples, then incubated at 37°C without shaking for 30 min and with shaking for an additional 30 min. Individual cultures were centrifuged for 15 min at 3850 rpm using a microcentrifuge (Micro12, Hanil). Centrifuged culture supernatants were removed for separation and replaced with 5 ml 2xYT medium containing 1 ml 1 M IPTG (AG Scientific), 5 ml 1 M MgCl ₂ , 1 ml 70 μg/μl kanamycin (Biopure), and 1 ml chloramphenicol. The resulting precipitate was thoroughly dispersed in the newly added medium, followed by incubation at 30°C for 16 hours with stirring.

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Table 9

F.A. M19 A5 Sequences

Dick

II

QFLKEGQLAAGTCEIVTLDRDSSQPRRTIARQTARCACRKGQIAGT TRARPACVDARIIKTKQWCDMLPCLEGEGCDLLINRSGWTCTQPG GRIKTTTVS (SEQ ID NO: 86) type

Ml

M2

M3

M4

M5

MB

M7

M8

OFLKEGOLAAGTCEVIAAHRDSSOPRRTIARQTARCACRKGOIAG TTRARPACVDARIIKTKQWCDMLPCLEGEGCDLLINRSGWTCTQP GGRIKTTTVS (SEQ ID NO: 87)

QFLKEGOLAAGTCEIVTLDRCCNKNRRTIARQTARCACRKGOIAG TTRARPACVDARIIKTKQWCDMLPCLEGEGCDLLINRSGWTCTQP GGRIKTTTVS (SEQ ID NO: 88)

QFLKEGQLAAGTCEIVTLDRDSSQPRIEERSQTARCACRKGQIAGT TRARPACVDARIIKTKQWCDMLPCLEGEGCDLLINRSGWTCTQPG GRIKTTTVS (SEQ ID NO: 51)

QFLKEGOLAAGTCEIVTLDRDSSOPRRTIARQTVKCSCFPGOIAGT TRARPACVDARIIKTKQWCDMLPCLEGEGCDLLINRSGWTCTQPG GRIKTTTVS (SEQ ID NO: 52)

QFLKEGQLAAGTCEIVTLDRDSSQPRRTIARQTARCACRKGQIAGT TRNKPSCVDARIIKTKQWCDMLPCLEGEGCDLLINRSGWTCTQPG GRIKTTTVS (SEQ ID NO: 53)

QFLKEGQLAAGTCEIVTLDRDSSQPRRTIARQTARCACRKGQIAGT TRARPACVDARIILQRWWCOMELCLEGEGCDLINRSGWTCTOP GGRIKTTTVS (SEQ ID NO: 54)

QFLKEGQLAAGTCEIVTLDRDSSQPRRTIARQTARCACRKGQIAGT TRARPACVDARIIKTKOWCDMLPCLEGEECKTLPDNSGWTCTQP GGRIKTTTVS (SEQ ID NO: 55)

QFLKEGQLAAGTCEIVTLDRDSSQPRRTIARQTARCACRKGQIAGT TRARPACVDARIIKTKQWCDMLPCLEGEGCDLLINRSGWTCSCSS GNKIKTTTVS (SEQ ID NO: 56)

As shown in FIG. 5, antibodies to FAM19A5 1-28 and 3-2 failed to bind to the FAM19A5 M2 mutant. As shown in table. 8, the M2 mutant has substitutions at amino acid residues 21-25 (ie, the EP2 epitope), which correspond to a region within the FAM19A5 F2 epitope fragment. This is consistent with earlier data from example 7 and confirms the importance of the F2 epitope fragment for the binding of antibodies 1-28 and 3-2 to FAM19A5.

As for antibody 2-13, it appears to bind to FAM19A5 at epitopes that are completely different from antibodies 1-28, 3-2 and 1-65. As shown in FIG. 5, the antibody to FAM19A5 2-13 had moderate to high binding to the FAM19A5 mutants M1-M3 and M5-M7, but did not bind to the M4 and M8 mutants, indicating the importance of the EP4 and EP8 epitopes in the binding of antibodies 2-13 to FAM19A5. Given that antibody 2-13 does not appear to bind to any of the FAM19A5 epitope fragments from Example 7 (see FIG. 2), these data also suggest that antibody 2-13 has a conformational epitope in counterbalance to the linear epitope.

Example 8. Competitiveness analysis.

A two-site sandwich ELISA was then used as described below to assess whether different anti-FAM19A5 antibodies compete with each other with similar epitope binding, see FIG. 6A.

First, the indicated antibodies to FAM19A5 were diluted in 1x PBS to a concentration of 10 μg/ml. Diluted anti-FAM19A5 antibody (10 µg/ml in 1x PBS) (capture antibody) was used to coat 96-well plates (100 µl/well) at 37°C for approximately 1 hour. After incubation, the plates were washed with wash buffer (0.01 % Tween-20/PBS; also called 0.01% PBST) with a total of five washes, blocked with blocking solution (5% BSA/PBS; also called 5% PBSA) (250 μl/well) for 1 h at 37°C and then washed again. 100 µg/ml FAM19A5 antigen (diluted in PBS containing 5% BSA, 0.01% Tween-20; also called 5% PBSAT; diluent buffer) was then added to each well and the 96-well plates were incubated for 2 h at 37°C. After incubation, the plates were washed using 0.01% PBST for a total of five washes. After the last wash, the indicated biotinylated anti-antibodies

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FAM19A5 (detection antibody) (diluted to 1 μg/ml in 5% PBSAT) was added to the appropriate wells (100 μl volume) and the plates were incubated for an additional 1 h at 37°C. The plates were then washed again with 0.01% PBST (five complete washes). Then, 100 μl of diluted (1/2000 in 5% PBSAT) streptavidin-HRP (1 mg/ml, Sigma, USA) was added to the wells and the plates were incubated for 30 min at room temperature. The plates were then washed and treated with 100 µl TMB substrate (3.3 plates then washed and treated with 100 µl solution, Thermo Fisher Scientific). After an additional 30 min incubation at room temperature, the color change reaction was induced by the addition of TMB substrate. This reaction was stopped using 50 μl of sulfuric acid (2N H ₂ SO ₄ ) and the extent of color change was determined by absorbance at 450 nm with a reference wavelength at 620 nm using a 96-well microplate reader (Molecular Device).

As shown in FIG. 6B, antibodies to FAM19A5 1-65, P2-A03, P2-P11 and 13B4 all cross-compete with each other. Like antibody 1-65, antibodies P2-A03, P2-F11, and 13B4 all bind to FAM19A5 at the M6, M7, and/or M8 epitopes (ie, within the F5 and/or F6 epitope fragments) (data not shown). In contrast, antibodies 2-13 and 3-2 did not cross-compete with other antibodies to FAM19A5, confirming earlier epitope mapping analysis that showed that these antibodies bind preferentially to the F2 epitope fragment.

Example 9 Binding Affinity Analysis

To determine the binding affinity of various anti-FAM19A5 antibodies to FAM19A5, an ELISA assay was used as described previously (eg, Example 6). As shown in FIG. 7A and 7B, antibodies 1-28, 3-2 and 1-65 bound to FAM19A5 with similar binding affinities (Kd = 0.17, 0.12 and 0.10 nM, respectively). Antibody 2-13 bound with a Kd value of approximately 2.77 nM, see FIG. 7B.

Example 10 Serum analysis for liver fibrosis.

To begin evaluating the in vivo function of the anti-FAM19A5 antibodies described herein, FAM19A5 protein levels in liver fibrosis were measured using a rat model of liver fibrosis (i.e., bile duct ligation (BLD)-induced Sprague-Dawley (SD) rat model of liver fibrosis ). See Kountouras et al., Br. J. Exp. Path. 65:305-311 (1984). Briefly, animals were anesthetized with ketamine and xylazine. A 1.5 cm midline incision was made and the common bile duct was located and ligated twice with 3-0 silk ligatures. Then, 21 days after induction of liver fibrosis, whole blood was collected from the animals and serum was separated from the whole blood.

Serum FAM19A5 protein levels were measured using the FAM19A5 sandwich ELISA. A 96-well ELISA plate was coated overnight with 1 μg/ml 1-65 human IgG1 anti-FAM19A5 antibody in a volume of 100 μl. The plate was then washed with PBS and then blocked with 1% BSA in PBS for 1 h at room temperature. The plate was then washed again with PBS and approximately 100 μl of standard and the sample was placed in the appropriate wells. The plate was then incubated at room temperature for 2 hours. The plate was then washed with 0.05% PBS-Tween 20 (i.e., wash buffer) and approximately 100 μl of detection antibody (horseradish peroxidase (HRP)-conjugated goat α-antibody). rabbit IgG (Santa Cruz, Cat #SC-2030) diluted 1:5000 in 1% BSA in PBS) was added to the wells. The plate was incubated at room temperature for an additional 1 hour. After additional washes with wash buffer, approximately 100 μl of O-phenylenediamine dihydrochloride (OPD) substrate was added to each well and the plate was allowed to develop at room temperature. After about 10 minutes, approximately 100 μl of 1N was added to the wells. H ₂ SO ₄ (sulfuric acid) to stop the reaction. Absorbance was immediately measured at 492 nM using a 96-well MicroPlate reader (Molecular Device, Versa Max).

As shown in FIG. 8, the level of FAM19A5 protein was more than 3-fold higher in the serum of SD-induced liver fibrosis rats (disease model) compared with the level in normal (i.e., non-BDL-induced) rats.

Example 11 Serum analysis for pulmonary fibrosis.

To evaluate whether FAM19A5 protein levels were also increased in pulmonary fibrosis in 7-week-old male SD rats, idiopathic pulmonary fibrosis was induced by administering 3 mg/kg bleomycin via intratracheal injection. 24 days after bleomycin administration, whole blood was collected from the animals and serum was separated from the whole blood. Serum FAM19A5 protein levels were measured using the FAM19A5 sandwich ELISA as shown in Example 10 above. As shown in FIG. 9, FAM19A5 protein level was more than 1.5-fold higher in the serum of SD rats with induced pulmonary fibrosis (disease model) compared with that in normal (i.e., without idiopathic pulmonary fibrosis) rats.

Example 12: Analysis of serum from patients with human liver fibrosis.

AST and/or ALT levels may increase in serum when hepatocytes are damaged, and such increases have often been suggested as liver damage. See Gowda et al., Pan. Afr. Med. J. 3:17 (2009); Giannini et al., Arch. Intern. Med. 163:218-224 (2003). However, when the damage is severe (eg, cirrhosis), AST and ALT levels may appear normal. Therefore, to evaluate whether FAM19A5 expression may be a better indicator of liver damage, FAM19A5 protein level

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FAM19A5 ELISA.

Human IgG1 antibody to FAM19A5 2-13 was reconstituted at a concentration of 1 μg/μl in 50 mM carbonate buffer (Bio-World, Cat. C2070-9.6, Lot. C70-9.616Z11C). Anti-FAM19A5 antibody was then diluted 1:1000 in PBS and then used to coat a 96-well ELISA plate overnight. The next day, the plate was washed with PBS and then blocked with 1% BSA in PBS at room temperature for approximately 1 hour. After this, the plate was washed again with PBS and approximately 100 μl of standards and samples were placed in the appropriate wells. The plate was incubated at room temperature for 2 hours. After this, the plate was washed with 0.05% PBS-Tween 20 (ie wash buffer). Then 3-2 mouse anti-FAM19A5 IgG2a antibody (reconstituted at 1 μg/μl) was diluted 1:2000 in 1% BSA in PBS. Approximately 100 μl of this diluted antibody was added to each of the wells and the plate was allowed to incubate for an additional 2 hours at room temperature. After an additional wash with wash buffer, approximately 100 μl of horseradish peroxidase (HRP)-conjugated donkey mouse IgG antibody (Jackson Lab, Cat. 715-035-151, Lot. 122401) (diluted 1:5000 in 1% BsA in PBS) was added into the holes. The plate was then incubated for 1 hour at room temperature. After incubation, the plate was washed with wash buffer and approximately 100 μl of 1-STEP™ Ultra TMB-ELISA substrate solution (Pierce, Cat. 34028, Lot. QJ20436540) was added to each well. After a 10-minute incubation, approximately 100 μl of 1N H ₂ SO ₄ (sulfuric acid) was added to the wells to stop the reaction. Absorbance was immediately measured at 450 nM using a 96-well MicroPlate reader (Molecular Device, Versa Max).

As shown in FIG. 10, patients with liver cirrhosis had FAM19A5 protein levels that were approximately 2-6 times higher than the levels observed in the serum of normal healthy individuals. Such data are consistent with the animal studies described above and indicate that serum FAM19A5 protein concentration may be an ideal diagnostic marker for liver cirrhosis.

Example 13. Analysis of the effect on myocardial infarction.

The death of ischemic cells that occurs during myocardial infarction leads to a reparative response in which the damaged tissue is replaced by a fibrous scar. This is followed by remodeling of the surrounding tissue (eg, thinning of the left ventricular wall and dilatation of the chamber), which ultimately leads to impaired cardiac function. See Talman et al., Cell Tissue Res. 365(3):563-581 (2016); Firth et al., Cardiovasc. Drugs Ther. 4:1363-1374 (1990). To evaluate the effect of the anti-FAM19A5 antibody on myocardial infarction, a mouse ischemic reperfusion model was used. Briefly, Balb/cAnNCrljOri mice (6 weeks old, male) (Orient Bio Inc., Seoul, South Korea) were purchased and maintained under specific pathogen-free conditions (SPF) for approximately 1 week. The mice were then deeply anesthetized with Zoletil 50 (VIRBAC, France) and xylazine (ROMPUN®, Bayer AG, Germany). After anesthesia, the mice underwent open thoracotomy to expose the coronary artery, and myocardial infarction was induced by ligating the artery using a 7-0 suture and PE20 tubing. In mice, forced breathing was maintained by connecting the endotracheal tube to a ventilator after air intubation. The ischemic state was maintained for 40 min, after which the blood was reperfused by removing the PE20 tube. Beginning on day 3 after induction of myocardial infarction, anti-FAM19A5 antibodies (1-65, 3-2, or 1-28 antibodies) were administered to mice via intravenous injection at weekly intervals for a total of three injections. Naïve (i.e., healthy) and NHI mice were used as controls.

On day 21 after induction of myocardial infarction, mice were sacrificed and their hearts were collected. Heart tissues were fixed in 10% formalin solution. Fixed tissues were then sectioned transversely at a thickness of 2 mm around the apex and embedded in paraffin blocks, which were then cut into 5-μm sections and mounted on glass slides for analysis. Hematoxylin and eosin (H&E) staining was performed on cardiac tissue samples to evaluate the effect of each anti-FAM19A5 antibody (i.e., 1-65, 3-2, and 1-28 antibodies) on tissue remodeling associated with myocardial infarction.

As expected, heart tissue from the NHI control group showed significant left ventricular dilatation and left ventricular wall thinning, see FIG. 11 (NHI). In contrast, heart tissue from mice that received antibodies to FAM19A5 resembled heart tissue from naïve (healthy) animals (i.e., reduced left ventricular dilatation and preserved left ventricular wall tissue).

To quantify this effect, Masson's trichrome, which stains collagen, was performed on heart tissue sections. As noted previously, collagen is a major component of the extracellular matrix (ECM), and therefore Masson's trichrome staining of increased collagen accumulation in a tissue sample can be an accurate indicator of fibrosis. Stained slides were photographed using an optical microscope (Olympus BX53, Japan), and the ratio of fibrous tissue area to total left ventricular area was also calculated using an image analyzer.

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As shown in FIG. 12A, mice that received antibodies to FAM19A5 (1-65, 3-2 and 1-28) had significantly less collagen accumulation compared to the NHI control group. In anti-FAM19A5 antibody treated groups, approximately 10-15% of the total left ventricular area was fibrous tissue, see FIG. 12V. In contrast, in NHI control mice, the fibrous area comprised approximately 20% of the total left ventricular area. Among the various FAM19A5 antibodies, antibody 1-65 had the greatest impact.

Taken together, the above results demonstrate that administration of FAM19A5 antibodies soon after myocardial infarction can significantly improve tissue remodeling and fibrosis formation.

Example 14: Analysis of Patients with Human Liver Cancer.

Liver fibrosis and cirrhosis are major risk factors for liver cancer. Patients with human liver cancer often have fibrosis, similar to cirrhosis, surrounding the cancerous tissue. Therefore, to evaluate FAM19A5 expression in liver cancer, FAM19A5 protein expression was measured in human liver biopsies using immunohistochemistry. Briefly, liver samples were obtained from liver cancer patients with varying degrees of fibrosis (i.e., stage 0 to stage 4). Tissue samples were immunostained with anti-FAM19A5 antibody and then stained with hematoxylin and eosin (H&E).

As shown in FIG. 13, there was a significant increase in FAM19A5 protein expression in the liver tissue of the patient with stages 2 and 4 compared with the patient with stage 0. The increased expression of FAM19A5 was centralized mainly around areas of scar formation and in hepatic stellate cells. Increased FAM19A5 expression also correlated with disease progression, with stage 4 liver tissue expressing much higher levels of FAM19A5 compared to stage 2 liver tissue. Taken together, these data demonstrate that FAM19A5 also plays an important role in human liver cancer.

Example 15. Analysis of the effectiveness of administering an antibody to FAM19A5 in a liver cancer xenograft model.

To evaluate the antitumor efficacy of antibodies to FAM19A5 in human liver cancer, a xenograft model of liver cancer in mice was used. Briefly, nude mice were purchased and housed in specific pathogen-free (SPF) conditions. After approximately a week of adaptation, the animals were injected subcutaneously with Hep3B cells and/or human hepatic stellate cells (HHSteC). To do this, cells were first treated with trypsin to prepare a single cell suspension. The cells were then washed and approximately 5 x 106 Hep3B cells and/or 0.4 x 106 HHSteC were resuspended in 100 µl DMEM. The cells were then injected subcutaneously into the right flank of nude mice using an insulin syringe. Approximately 3 weeks after injection, animals were observed for tumor formation.

After tumor formation (∼3 weeks postinjection), animals were injected intravenously with either normal human immunoglobulin (NHI, control) or 3-2 human IgG1 anti-FAM19A5 antibodies (2.5 mg/kg). Animals received antibodies once a week for 3 weeks. Body weight and tumor size were assessed every week. And 42 days after inoculation, the animals were sacrificed and the tumors were subjected to further analysis.

As shown in FIG. 14A, animals from all different groups had the same body weight throughout the entire experimental period. And, as shown in FIG. 14B-14D, administration of antibody to FAM19A5 had minimal antitumor effect in animals inoculated with Hep3B cells only (ie, Hep3B + NHI and Hep3B + FAM19A5 Ab). But in animals inoculated with both Hep3B and HHSteC cells, administration of the FAM19A5 antibody resulted in a significant reduction in tumor size/weight (i.e., Hep3B + HHSteC + NHI vs. Hep3B + HHSteC + FAM19A5 Ab). Such data indicate that the interaction of anti-FAM19A5 antibody with hepatocellular stellate cells in the tumor microenvironment can treat liver cancer.

Example 16. Effect of administration of an antibody to FAM19A5 after traumatic brain injury.

To evaluate the effect of the anti-FAM19A5 antibodies described herein on traumatic brain injury, a model of traumatic brain injury (TBI) was used. Briefly, adult male C57BL/6 mice (8–9 weeks old) were deeply anesthetized with isofluorane. Cryogenic TBI was performed by placing an iron rod (pre-cooled using liquid nitrogen) on the jaw for 1 min. The mouse's skin was then sutured and maintained in the same manner as normal mice. Moon et al., Neuro Report. 22:304-308 (2011). Approximately 1 day after induction of TBI, animals were intravenously injected with 100 μg of various anti-FAM19A5 antibodies (1-65 (2/4); 1-28; 2-13 and 3-2) diluted in phosphate-buffered saline. (PBS).

At day 5 post-TBI induction (TBI5D), animals were sacrificed, perfused with 4% paraformaldehyde (PFA) in PBS, and brain tissues were collected. The collected brain tissues were further fixed in 4% PFA solution for an additional 24 h.

Brain tissues were then cytoprotected in 30% sucrose, serially sectioned on a cryostat (40 μm), and stored in 50% glycerol/50% PBS at −20°C until use.

To stain reactive astrocytes associated with gliosis (positive for expression

- 62 043381 nestin and GFAP), brain tissue sections were blocked with 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS for 30 min. Primary antibodies were then incubated with sections overnight at 4°C. The primary antibodies used in this study were mouse anti-nestin (Millipore, Billerica, MA, USA) and rabbit anti-GFAP (Dako, Carpinteria, CA, USA). After several washes with PBS, the appropriate secondary antibodies were applied for 30 min. Nuclei were labeled with Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). Sections were then washed, mounted, and observed under a fluorescence or confocal microscope (Leica, Wetzlar, Germany).

To quantify the effect, the region of interest (GI)-negative region for each image was first specified, and then the area ( ^µm2 , A) of the corresponding ROI was calculated using LAS AF lite software (Leica Microsystem CMS GmbH, Mannheim, Germany). The lateral length (µm, V) of the injury core line contacting the penumbra was also measured using the same software. The A/B result indicates the average distance from the lesion core (A/B, µm).

As shown in FIG. 15A and 15B, antibodies 1-28, 2-13 and 3-2 to FAM19A5 similarly reduced, eliminated and/or prevented the occurrence of reactive gliosis in the penumbral regions after traumatic brain injury. The overall effect was similar to that of the anti-FAM19A5 antibody 1-65, which has previously been shown to significantly attenuate the formation of reactive astrocytes after traumatic brain injury. See US Patent No. 9579398. Although FAM19A5 protein binds to a different epitope than antibody 1-65, antibodies 1-28, 2-13 and 3-2 were equally effective when administered after traumatic brain injury.

Example 17. Evaluation of the antitumor effect of the antibody FAM19A5 in the mouse glioblastoma cell line of cancer cells GL-261.

To induce the head glioblastoma model, a 4-week-old C57BL/6 mouse was fixed in a stereotaxic apparatus, and GL-261 cells (1x105 cells/5 μl) were injected intracranially (intracranial injection: 0.2 mm anterior to bregma, 2.2 mm lateral to the midline , 3.5 mm depth from the surface of the skull to the middle of the caudal part of the abdominal cavity) to induce gyoblastoma (Fig. 16A). Human IgG (negative control) and anti-FAM19A5 antibody were administered intravenously seven days after GL-261 inoculation into the respective animals. Each mouse received either human IgG control antibody or anti-FAM19A5 antibody at a dose of 2.5 mg/kg once a week, three times (Fig. 16A).

Beginning 23 days after GL-261 inoculation, death occurred and, therefore, all surviving mice were sacrificed and the brains were removed and fixed with phosphate-buffered saline containing 4% paraformaldehyde. To measure tumor size in brain tissue, three analytical methods were used: 1) tissue clearing (CLARITY) (Fig. 16B), 2) hematoxylin and eosin (H&E) staining (Figs. 17A and 17B), and 3) Hoechst (Figs. 18A-18D).

(1) For tissue transparency, the assay tissue was first immersed in a hydrogel monomer solution (A4P0) and stored at 4 °C for one day to ensure penetration of the hydrogel throughout the tissue. Thereafter, the temperature was raised to 37°C to induce polymerization to form a network structure. Electrophoresis tissue clearing (ETC) was then used to remove lipids from the tissue. The prepared tissue was then placed in a lightbox. The surface of the relatively opaque tissue was considered as the surface of the tumor, and the approximate tumor weight was measured using a scale.

(2) Hematoxylin and eosin staining (H&E stain) after fixation with phosphate-buffered saline containing 4% paraformaldehyde for 24 h and then suspension in phosphate-buffered saline containing 30% sucrose for another 24 h. The tissue was then placed into a brain tissue mold, and the OCT compound containing 30% sugar solution was frozen on dry ice, and the brain tissue was thinly sliced to 30 μm using a cryostat microtome (Leica). After completion of H&E staining, images of the prepared sample were acquired using a slide scanner (Carl Zeiss/AxioScanZ) and the tumor boundary surface was randomly determined using a ZEN microscope and imaging software (Zeiss) to obtain the tumor surface area value.

(3) Thinly cut tissue for Hoechst staining was treated with phosphate buffer solution containing Hoechst (1:2000) for 30 min. The fluorescence image of the prepared sample was obtained using a confocal microscope (Leica), and the tumor boundary was determined using IMARIS 3D software (Bitplane) to obtain the tumor volume and number of cells in the tumor. As shown in FIG. 16B, 17A, 17B, and 18A-18D, measurement of tumor size in each mouse showed that the size of glioblastoma in the anti-FAM19A5 antibody-treated group was significantly reduced compared with the human IgG-treated group in all three experimental methods.

Example 18. Promotion of vascular normalization after treatment with the FAM19A5 antibody in a mouse model of glioblastoma.

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Brains were prepared by killing glioblastoma-induced mouse models (see Example 17) after fixation with phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 24 h, followed by infiltration in PBS containing 30% sucrose for an additional 24 hours The brain was then placed in a brain tissue mold and frozen on dry ice using OCT compound containing 30% sugar solution. The frozen brain was then thinly sliced to 30 μm using a cryostat microtome. Immunohistochemistry was performed to determine the protein expression pattern of CD31 (a marker of vascular endothelial cells) in glioblastoma-induced mouse brain. Fluorescence images of the prepared sample were obtained using a confocal microscope (Leica).

When comparable surface areas were examined (Fig. 19A, indicated by white dots), in the control group the number of blood vessels and their connectivity were very sparse (Fig. 19A, left column), whereas in the anti-FAM19A5 antibody-treated group, the number of blood vessels and their reporting were relatively high (Fig. 19A, right column). See fig. 19B for a higher magnification of the characteristic region in FIG. 19A.

The above results showed that treatment with anti-FAM19A5 antibody could promote the normalization of blood vessels, which could facilitate the treatment of glioblastoma.

Example 19: Differences in immune cell distribution following treatment with FAM19A5 antibody in a mouse model of glioblastoma.

Brains isolated from glioblastoma-induced mouse models (see Example 17) were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 24 hours, followed by permeation in PBS containing 30% sucrose for an additional 24 hours. The brain was then placed in a brain tissue mold and frozen on dry ice using OCT compound containing a 30% sugar solution. The frozen brain was then thinly sliced to 30 μm using a cryostat microtome. The distribution of astrocytes and macrophages in glioblastoma was observed by performing immunohistochemistry for GFAP (astrocyte marker) and Iba-1 (macrophage marker).

In the control group, no astrocytes were observed in the glioblastoma, whereas in the FAM19A5 antibody-treated group, relatively large numbers of astrocytes remained in the tumor. Astrocytes in the tumor are expected to have a positive effect on peripheral neurons or the extracellular environment of neurons. In addition, analysis of the level of macrophage infiltration in glioblastoma showed that macrophage infiltration increased dramatically in the anti-FAM19A5 antibody-treated group compared with the human IgG negative control treated group (FIGS. 20A and 20B).

These results indicated that anti-FAM19A5 antibody treatment could also promote macrophage infiltration into glioblastoma, which could then facilitate treatment of glioblastoma.

Example 20. Evaluation of the anticancer efficacy of the FAM19A5 antibody in a rat glioma model using C6 tumor cells.

To induce an isogenic rat glioma model, 1.8x105/1 μl C6 tumor cells were inoculated into male Wistar rats by intracranial injection using a stereotaxic frame. From day 7 after tumor inoculation, each rat was administered intravenously with human IgG (negative control) antibody or anti-FAM19A5 antibody at a dose of 2.5 mg/kg every three days for two times. MRI scan image to analyze the inoculated tumor size, location and qualitative results of each rat was obtained 2-3 days after the 7th day after tumor cell inoculation.

T2-weighted image scanning is a basic morphological analysis that provides a visual and qualitative assessment of the location and size of the inoculated tumor, etc. R2 mapping analysis, used as an indicator of fluid content of tumor tissue, allows visual confirmation of the amount of edema in the tumor. The visual analytical method used to determine total blood flow was used to determine the change in R2* value before and after administration of high molecular weight T2 contrast agent MION (size: 20±5 nm, dose: 5 mg/kg), which calculated CBV (cerebral blood volume) via delR2* mapping. The CBV (cerebral blood volume) value was determined by the variation of R2 and used for visual analysis of the total blood volume in the brain tissue. The rate of improvement (EnhRate) and area under the curve (AUC) were representative parameters to determine tissue permeability.

As shown by the scanned image (FIG. 21), T2-weighted image showed that administration of anti-FAM19A5 antibody effectively inhibited tumor growth compared with human IgG antibody (negative control). The result of R2 mapping in animals treated with anti-FAM19A5 showed a decrease in the area of necrosis in the tumor, which resulted in a decrease in water content in the tumor core compared to animals treated with human IgG (negative control). The results of the CBV assay showed a relatively large volume of blood in the tumor tissue of animals treated with anti-FAM19A5 compared to animals treated with human IgG (negative control), demonstrating an increase in blood flow rate in the tumor tissue. Finally, an assessment of the permeability of tumor tissue showed a decrease in permeability in

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Claims (11)

animals treated with anti-FAM19A5 antibody compared with animals treated with human IgG (negative control). IAUC30 denotes the initial area under the curve after 30 s. These results, obtained in a rat C6 glioma tumor cell model treated with anti-FAM19A5 antibody, compared with a negative control group treated with human IgG, show a decrease in the area of necrosis, leading to attenuation of edema. Effective suppression of tumor growth occurred by reducing the permeability of tumor cells and improving blood flow velocity. Example 21. Evaluation of the anticancer efficacy of the antibody to FAM19A5 in a mouse brain cancer model using GL261 tumor cells. To further evaluate the antitumor effect of the anti-FAM19A5 antibody, GL261 Red-FLuc cells were used to induce brain cancer in mice. Briefly, GL261 Red-FLuc cells (3x105) were injected into the brains of C57BL/6 mice (day 0). Mice were then intravenously injected with human IgG (Sigma, cat. #14506) or anti-FAM19A5 antibody (clone 3-2, Lot #171123) 3 days after injection. Antibodies were administered at a dose of 5 mg/kg once a week for a total of four weeks. After the last antibody administration (day 24), mice were monitored for survival until all animals died. As shown in FIG. 22, in the human IgG group, the last mouse died on day 28 after injection of GL261 cells. In contrast, in animals treated with anti-FAM19A5 antibody, the last mouse died a week later (i.e., on day 35 after injection of GL261 cells). This result further confirmed the anti-cancer efficacy of anti-FAM19A5 antibody, demonstrating that anti-FAM19A5 antibody can improve survival in animals with brain cancer. It should be noted that the Detailed Description section, and not the Summary and Abstract sections, is intended to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, exemplary embodiments of the present specification as contemplated by the inventor(s), and thus are not intended to limit the present specification and the appended claims in any way. The present invention has been described above using functional building blocks illustrating the implementation of certain functions and their relationships. The boundaries of these functional building blocks have been arbitrarily defined herein for ease of description. Alternative boundaries may be defined provided that the specified functions and their relationships are properly performed. The foregoing description of specific embodiments will represent the general nature of the specification as fully as others can, using knowledge of the art, readily modify and/or adapt for various applications specific embodiments without undue experimentation without departing from the general concept of the present specification. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the described embodiments based on the instructions and guidance provided herein. It should be understood that the phraseology or phraseology herein is intended for purposes of description and not limitation, so that the terminology or phraseology herein should be interpreted by one skilled in the art in light of the instructions and guidelines. The breadth and scope of the present specification is not to be limited by any of the exemplary embodiments described above, but is to be defined only by the following claims and their equivalents. All publications, patents, patent applications, Internet sites and database accession numbers/sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes at the same degree as if each individual publication, patent, patent application, Internet site or accession number/database sequence were specifically and individually designated for incorporation by reference. The PCT application for this invention claims priority from US Provisional Application No. 62/525635, filed June 27, 2017, and 62/582,887, filed November 7, 2017, both of which are incorporated herein by reference in their entirety. CLAIM 1. An isolated antibody or antigen-binding portion thereof that specifically binds to the human family with sequence similarity 19 (FAM19A5) member A5 protein (anti-FAM19A5 antibody), and which contains a heavy chain variable region (VH) containing heavy chain CDR1, CDR2 and CDR3 chain, and a light chain variable region (VL) comprising light chain CDR1, CDR2 and CDR3, wherein: a) said VH contains an amino acid sequence that is at least 85% - 65 043381 is identical to the amino acid sequence presented in SEQ ID NO: 36, and the specified VL contains an amino acid sequence that is at least 85% identical to the amino acid sequence presented in SEQ ID NO: 40; b) said heavy chain CDR3 contains the amino acid sequence shown in SEQ ID NO: 16; And c) said anti-FAM19A5 antibody is capable of binding the FAM19A5 epitope set forth in SEQ ID NO: 6. 2. Antibody to FAM19A5 according to claim 1, characterized in that the heavy chain CDR1 contains the amino acid sequence presented in SEQ ID NO: 14, the heavy chain CDR2 contains the amino acid sequence presented in SEQ ID NO: 15, the heavy chain CDR3 contains the amino acid sequence set forth in SEQ ID NO: 16, light chain CDR1 contains the amino acid sequence set forth in SEQ ID NO: 26, light chain CDR2 contains the amino acid sequence set forth in SEQ ID NO: 27, and light chain CDR3 contains the amino acid sequence set forth in SEQ ID NO: 27 ID NO: 28. 3. Antibody to FAM19A5 according to claim 1 or 2, characterized in that said VH contains the amino acid sequence set forth in SEQ ID NO: 36, and said VL contains the amino acid sequence set forth in SEQ ID NO: 40. 4. Antibody to FAM19A5 according to any one of claims 1 to 3, characterized in that said antibody is a chimeric antibody, a human antibody or a humanized antibody. 5. Antibody to FAM19A5 according to any one of claims 1 to 4, characterized in that said antibody to FAM19A5 contains a heavy chain containing the amino acid sequence presented in SEQ ID NO: 58, and a light chain containing the amino acid sequence presented in SEQ ID NO: 62. 6. Antibody to FAM19A5 according to any one of claims 1-5, characterized in that said antibody has one or more of the following properties: (a) reduces, eliminates, delays and/or prevents fibrosis; (b) reduces the formation of excess extracellular matrix (ECM); (c) delays tumor growth or progression; (d) binds to soluble human FAM19A5 with a K _D of 10 nM or less, as measured by enzyme-linked immunosorbent assay (ELISA); (e) binds to membrane-bound human FAM19A5 with a K _D of 10 nM or less, as measured by ELISA; (f) reduces, eliminates, delays and/or prevents the development of reactive gliosis; (g) suppresses excessive proliferation of reactive astrocytes; (h) reduces the expression of chondroitin sulfate proteoglycans, including neurocan and neuroglial antigen 2 (NG2); (i) increases the expression of c-fos and pERK in neuronal nuclei; (j) promotes neuronal survival; (k) increases GAP43 expression in neurons and (l) promotes axon outgrowth. 7. Nucleic acid encoding an antibody to FAM19A5 according to any one of claims 1 to 6. 8. A composition for modulating the activity of FAM19A5, containing an antibody to FAM19A5 according to any of claims 1-6 or a nucleic acid according to claim 7 and a carrier. 9. A pharmaceutical composition containing an antibody to FAM19A5 according to any of claims 1-6 or a nucleic acid according to claim 7 and a carrier. 10. A method of treating brain cancer in a subject in need thereof, comprising administering to said subject an anti-FAM19A5 antibody according to any one of claims 1 to 6, a nucleic acid according to claim 7, a composition according to claim 8, or a pharmaceutical composition according to claim 9. 11. A method for diagnosing fibrosis and/or cancer of the brain in a subject in need thereof, comprising contacting a biological sample of said subject with an antibody to FAM19A5 according to any one of claims 1 to 6, to determine the level of FAM19A5 protein in said biological sample, when wherein said subject has fibrosis and/or brain cancer or is at risk of developing fibrosis and/or brain cancer if the level of FAM19A5 protein in said biological sample is higher than the corresponding level in a biological sample from a subject who does not have fibrosis and/or brain cancer /or brain cancer and there is no risk of developing fibrosis and/or brain cancer. -