WO2025247171A1

WO2025247171A1 - Anti-mesothelin antibodies

Info

Publication number: WO2025247171A1
Application number: PCT/CN2025/097272
Authority: WO
Inventors: Franklin Grosveld; Marinus VAN HAPEREN; Dubravka Drabek; Richard JANSSENS; Meihong ZHANG; Changjing DENG; Liang Zhou; Lei Shi; Yun Zhang; Yuexiang YIN
Original assignee: Nona Biosciences Suzhou Co Ltd; Harbour Antibodies BV
Current assignee: Nona Biosciences Suzhou Co Ltd; Harbour Antibodies BV
Priority date: 2024-05-27
Filing date: 2025-05-26
Publication date: 2025-12-04
Anticipated expiration: 2026-11-27

Abstract

The invention provides antibodies and antigen-binding fragments thereof that recognize mesothelin (MSLN). In some embodiments, the antibodies bind to membrane-bound MSLN, but not to soluble MSLN. In some embodiments, the antibodies block the interaction between MSLN and MUC16. In some embodiments, the antibodies provide a means of treating mesothelin-positive cancer. In some embodiments, the antibodies are used to diagnose or image mesothelin-positive cancer.

Description

ANTI-MESOTHELIN ANTIBODIES

The invention claims the priority of PCT Application No. PCT/CN2024/095555 and European Application No. EP24178162.4, all of which are incorporated herein by reference in entirety.

FIELD OF THE INVENTION

The invention relates to antibodies and antigen-binding fragments thereof that bind to mesothelin (MSLN) .

BACKGROUND OF THE INVENTION

Carcinomas like mesotheliomas, pancreatic adenocarcinomas, ovarian cancers and lung adenocarcinomas are highly destructive and very difficult to treat diseases. For example, pancreatic ductal adenocarcinoma accounts for 90%of all pancreatic tumors and its incidence is rising while it has a very poor prognosis. The lack of available specific diagnostics tests and the very limited treatment opportunities present a serious health problem.

Mesothelin (MSLN) is a cell surface molecule that is expressed as a 71kD precursor protein which is further processed to a 40kD glycoprotein that is glycosylphosphatidylinositol (GPI) -anchored on the cell surface. MSLN shows a very limited and low expression in normal tissues. It is expressed in mesothelial cells that line the pleura, pericardium and peritoneum, where it appears to play a role in cell adhesion (Chang et al. (1996) PNAS 93: 136-140) . Soluble cleaved MSLN has also been proposed to play a role in megakaryocyte stimulation, but a knockout in mice did not show any defects in development and its biological role is therefore not clear (Yamaguchi et al. (1994) 269 (2) : 805-8; Bera et al. (2000) 20 (8) : 2902-6) . In contrast, MSLN is highly expressed in several human cancers, including virtually all mesotheliomas and pancreatic adenocarcinomas, and approximately 70%of ovarian cancers and 50%of lung adenocarcinomas (Hassan and Ho (2008) Eur. J. Cancer 44: 46-53; Miettinen and Sarlomo-Rikala (2003) Am. J. Surg. Pathol. 27: 150-8; Ordonez (2003) Am. J. Surg. Pathol. 27: 1418-1428; Ho et al. (2007) Clin. Cancer Res. 13: 1571-5) . Its high level of expression makes MSLN an attractive candidate for targeted therapy, because it plays an important role in tumour promoting proliferation and invasion (Servais et al. (2012) Clin. Cancer Res. 18 (9) : 2478-2489)

MSLN interacts with MUC16 mediating cell adhesion, which plays an important role in ovarian cancer cell peritoneal implantation and increases the motility and invasion of pancreatic carcinoma cells (Rump et al. (2004) J Biol Chem. 279 (10) : 9190-8; Gubbels et al. (2006) Mol Cancer 5 (1) : 50; Coehlo et al. Expert Rev Anticancer Ther. 18 (2) : 177-186; Chen et al. (2013) Sci Rep. 3: 1870) . Particularly pancreatic tumours have often progressed too far before patients feels any symptoms of the disease and the average survival time is short (often less than one year) . This prognosis is bad because the tumour can often not be (completely) removed surgically and has already (often undetectably) metastasized. Chemotherapy also does not lead a substantial improvement of survival time or cure. Despite several attempts there is as yet there also no successful immunotherapy. Even the best known monoclonal antibody against mesothelin (Amatuximab) , which binds the soluble form of MSLN is not particularly successful (Baldo and Cecco (2017) Onco. Targets Ther. 10: 5337-5353; Nicolaides et al. (2018) Cancer Biology &Therapy 19 (7) : 622-630) . Unfortunately the shedding of mesothelin by the tumor causes a number of problems, although it can be used as a biomarker for disease (Hassan et al. (2006) Clin. Cancer Res. 12: 447-453) . The shedding causes problems for the imaging of tumours and potential radiotherapy. The MSLN shedding into the bloodstream causes a high background for radiolabeled antibody based imaging and is toxic because a radiolabeled antibody binds the shed mesothelin throughout the body. This necessitates the administration of high and hence toxic doses of anti-mesothelin antibodies. Moreover its binding to MUC16 suppresses the immune effector function of anti-MSLN antibodies (Nicolaides et al. (2018) Cancer Biology &Therapy 19 (7) : 622-630) .

Monoclonal antibodies or variants thereof represent a high proportion of new medicines launched in this century. Monoclonal antibody therapy is already accepted as a preferred route for the treatment of a number of diseases such as rheumatoid arthritis, Crohn’s disease and there is impressive array of antibody based treatments of cancer. Antibody-based therapeutic products are also in development for other diseases such as cardiovascular and infectious diseases.

Two problems can arise in raising antibodies against a particular antigen when:

1. The antigen used for the immunisation is not in its normal configuration or when only part of the antigen can be used (e.g. in the case that the complete antigen protein is insoluble) . Antibodies are obtained against the antigen, but these do not recognize the antigen in its natural/native configuration.

2. The antigen of interest is very similar (e.g. homologous) or identical to a protein that is endogenously expressed in the host to be immunised. The immunized antigen may not be recognized as a foreign protein against which an immune response should be generated.

The issue of tolerance can in principle be circumvented by employing display methods using libraries of antibodies or binding domains to raise antibodies or VH/VHH domains directed against the antigen, since such methods are ex vivo. However, display methods have the disadvantage that they are cumbersome. In addition, in the case of HCAb, display methods often yield insoluble antibodies. In the case of tetrameric antibodies, display methods typically use a common light chain to ensure solubility for the heavy chain and, disadvantageously, a second screen or mutation optimisation is then required to obtain an optimal light chain.

In the case of cell surface antigens, these are usually screened in display methods with antigen peptide (s) or protein rather than cells with the antigen in its normal configuration on the cell surface, and the obtained antibodies may not recognize the antigen expressed on the cell surface.

Therefore, there is a need for improved methods for obtaining antibodies, particularly antibodies that specifically bind to: (1) antigens that are similar or identical to endogenous proteins in animals that are typically used for antibody generation or (2) antigens that have a complex or insoluble native configuration or (3) a complex of proteins (e.g. TGFβ precursor bound to GARP; Cuende et al. (2015) Science Translational Medicine 7 (284: 284ra56) ; Sheridan (2015) Nature Biotechnology 33 (7) : 673-675) .

SUMMARY OF THE INVENTION

The invention provides an anti-mesothelin antibody that binds to membrane bound mesothelin (MSLN) , the antibody does not bind to soluble MSLN.

The invention further provides an anti-mesothelin antibody that blocks the interaction between mesothelin (MSLN) and MUC16.

The invention further provides a combination of antibodies comprising: (i) an anti-mesothelin antibody that binds to membrane bound mesothelin (MSLN) , the antibody does not bind to soluble MSLN and (ii) an anti-MSLN antibody that blocks the interaction between MSLN and MUC16.

The invention further provides an isolated nucleic acid encoding an antibody of the invention.

The invention further provides a vector comprising a nucleic acid of the invention.

The invention further provides a host cell comprising a vector of the invention.

The invention further provides a pharmaceutical composition comprising the antibody, or combination of antibodies, of the invention and a pharmaceutically acceptable carrier.

The invention further provides an antibody of the invention for use in therapy.

The invention further provides an antibody of the invention for use in treating mesothelin-positive cancer.

The invention further provides a method for diagnosing mesothelin-positive cancer in a subject comprising:

(a) obtaining a biological sample from the subject,

(b) contacting the sample with an antibody of the invention, and

(c) detecting binding of the antibody to the sample,

an increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having a mesothelin-positive cancer.

The invention further provides a method for imaging a mesothelin-positive cancer in a subject comprising:

(a) administering an antibody of the invention to the subject, the antibody is conjugated to a detectable marker, and

(b) detecting the presence of the marker.

The invention further provides a method for producing an antigen-specific antibody in a transgenic non-human animal comprising:

(A) inducing expression of the antigen in the animal, and

(B) isolating antibodies that specifically bind to the antigen or cells that produce antibody that specifically binds to the antigen;

wherein step (A) is performed after the animal has established central tolerance.

The invention further provides a method for producing an antigen-specific antibody in a non-human animal comprising:

(a) reducing or eliminating expression of a protein that is similar or identical to the antigen in the animal,

(b) immunising the animal with the antigen, and

(c) isolating antibodies that specifically bind to the antigen or cells that produce antibody that specifically binds to the antigen;

wherein step (a) is performed before or during the period in which the animal is establishing central tolerance.

The invention further provides a method of producing an antigen-specific antibody in a non-human animal comprising:

(a) replacing an endogenous gene encoding a protein that is similar or identical to the antigen in the animal with a gene encoding a replacement protein that has a similar function to the endogenous protein but is sufficiently antigenically different from the antigen to avoid the animal being tolerised against the antigen,

(b) immunising the animal with the antigen, and

The invention further provides an antigen-specific antibody obtained by the production methods according to the invention.

In a first aspect, the invention provides an antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH) , and wherein

(1) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 212, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 226;

(2) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 215, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 226;

(3) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 213, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 227;

(4) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 194, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 220;

(5) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 199, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 220;

(6) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 195, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 221;

(7) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 195, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 225;

(8) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 196, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 222;

(9) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 200, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 222;

(10) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 197, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 223;

(11) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 198, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 224; or

(12) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 201, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 224.

In some embodiments, the invention provides an antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH) , and wherein

(1) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 36, 74, 121 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 149, 161, 177 respectively;

(2) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 37, 75, 122 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 150, 162, 178 respectively;

(3) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 27, 62, 107 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 144, 157, 171 respectively;

(4) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 27, 62, 112 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 144, 157, 171 respectively;

(5) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28, 63, 108 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 145, 158, 172 respectively;

(6) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28, 63, 108 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 145, 158, 176 respectively;

(7) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 29, 64, 109 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 146, 159, 173 respectively;

(8) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 29, 67, 113 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 146, 159, 173 respectively;

(9) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 30, 65, 110 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 147, 160, 175 respectively;

(10) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 31, 66, 111 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 148, 158, 174 respectively; or

(11) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 31, 66, 114 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 148, 158, 174 respectively,

wherein the CDRs are determined by EU Kabat system.

(1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 212, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 226;

(2) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 215, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 226;

(3) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 213, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 227;

(4) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 194, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 220;

(5) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 199, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 220;

(6) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 195, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 221;

(7) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 195, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 225;

(8) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 196, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 222;

(9) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 200, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 222;

(10) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 197, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 223;

(11) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 198, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 224; or

(12) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 201, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 224.

In some embodiments, the invention provides an antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) , and wherein:

(1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 259, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 273;

(2) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 262, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 273;

(3) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 260, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 274;

(4) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 238, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 267;

(5) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 245, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 267;

(6) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 240, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 268;

(7) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 240, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 272;

(8) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 241, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 269;

(9) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 246, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 269;

(10) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 243, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 270;

(11) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 244, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 271; or

(12) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 247, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 271.

In a second aspect, the invention provides an antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) , and wherein

(1) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 209;

(2) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 214;

(3) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 204;

(4) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 203;

(5) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 185;

(6) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 211;

(7) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 216;

(8) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 217;

(9) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 218;

(10) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 202;

(11) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 206;

(12) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 205;

(13) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 207;

(14) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 208;

(15) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 210;

(16) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 186;

(17) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 187;

(18) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 188;

(19) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 189;

(20) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 190;

(21) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 191;

(22) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 192; or

(23) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 193.

In some embodiments, the invention provides an antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) , and wherein

(1) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 35, 70, 117 respectively;

(2) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 38, 76, 123 respectively;

(3) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 27, 69, 116 respectively;

(4) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 27, 68, 115 respectively;

(5) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 22, 56, 100 respectively;

(6) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 23, 58, 102 respectively;

(7) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 39, 77, 124 respectively;

(8) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 35, 73, 117 respectively;

(9) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 35, 71, 120 respectively;

(10) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 24, 58, 102 respectively;

(11) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 33, 71, 118 respectively;

(12) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 32, 70, 117 respectively;

(13) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 34, 72, 119 respectively;

(14) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 33, 71, 120 respectively;

(15) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 35, 73, 120 respectively;

(16) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 23, 57, 101 respectively;

(17) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 25, 56, 100 respectively;

(18) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 23, 59, 103 respectively;

(19) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 23, 60, 104 respectively;

(20) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 26, 61, 105 respectively; or

(21) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 22, 56, 106 respectively,

wherein the CDRs are determined by EU Kabat system.

(1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 209;

(2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 214;

(3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 204;

(4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 203;

(5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 185;

(6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 211;

(7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 216;

(8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 217;

(9) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 218;

(10) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 202;

(11) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 206;

(12) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 205;

(13) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 207;

(14) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 208;

(15) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 210;

(16) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 186;

(17) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 187;

(18) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 188;

(19) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 189;

(20) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 190;

(21) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 191;

(22) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 192; or

(23) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 193.

In some embodiments, the invention provides an antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a heavy chain (HC) , and wherein

(1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 256;

(2) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 261;

(3) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 251;

(4) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 250;

(5) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 249;

(6) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 258;

(7) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 263;

(8) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 264;

(9) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 265;

(10) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 248;

(11) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 253;

(12) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 252;

(13) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 254;

(14) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 255;

(15) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 257;

(16) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 229;

(17) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 230;

(18) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 231;

(19) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 232;

(20) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 233;

(21) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 234;

(22) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 235;

(23) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 236;

(24) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 237;

(25) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 275; or

(26) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 276.

In some preferred embodiments, the antibody does not comprise a light chain. In some more preferred embodiments, the antibody comprises two heavy chains.

In some preferred embodiments of the first aspect and the second aspect, the antibody or antigen binding fragment thereof specifically binds to membrane bound mesothelin. In some preferred embodiments, the antibody or the antigen binding fragment thereof binds to membrane bound mesothelin with a higher affinity as compared to the affinity of its binding to soluble mesothelin. In some preferred embodiments, the antibody or the antigen binding fragment thereof binds to membrane bound mesothelin with an affinity which is at least two folds, at least three folds, at least five folds, at least 10 folds, at least 20 folds, at least 30 folds, at least 50 folds, or at least 100 folds of the affinity of its binding to soluble mesothelin. In some preferred embodiments, the antibody or the antigen binding fragment thereof does not bind to soluble MSLN.

In some preferred embodiments of the first aspect and the second aspect, the antibody or the antigen binding fragment thereof blocks the interaction between MSLN and MUC16.

In some preferred embodiments of the first aspect and the second aspect, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

In some preferred embodiments of the first aspect and the second aspect, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD. In more preferred embodiments, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

In some preferred embodiments of the first aspect and the second aspect, the antigen binding fragment is selected from the group consisting of Fab, Fab’ , F (ab') ₂, Fd, Fd’ , Fv, scFv, ds-scFv and dAb.

In some preferred embodiments of the first aspect and the second aspect, the antibody is a monoclonal antibody, a bi-specific or a multi-specific antibody.

In some preferred embodiments of the first aspect and the second aspect, the antibody is monovalent, bivalent or multivalent.

In some preferred embodiments of the first aspect and the second aspect, the antibody or antigen binding fragment is attached to a fluorescent label, radiolabel or cytotoxic agent.

In some preferred embodiments of the first aspect and the second aspect, the antibody or antigen binding fragment is obtained using a transgenic non-human animal with human MSLN transgene by a method comprising:

(A) inducing expression of human MSLN in the animal, and

(B) isolating antibodies that specifically bind to MSLN or cells that produce antibody that specifically binds to MSLN;

In some embodiments, the method further comprises:

(A’) reducing or eliminating expression of endogenous MSLN in the animal,

wherein step (A’) is performed preferably before or during the period in which the animal is establishing central tolerance.

In some preferred embodiments, the animal is a rodent, optionally a mouse.

In some preferred embodiments:

(i) step (A’) is performed before or during the 1-2 weeks after birth of the animal, during the ten days after birth of the animal or for the lifetime of the animal, and/or

(ii) step (A) is performed later than eight days after birth.

In some preferred embodiments of the first aspect and the second aspect, the antibody is obtained using a non-human animal by the method comprising:

(a) reducing or eliminating expression of endogenous MSLN in the animal,

(b) immunising the animal with human MSLN, and

(c) isolating antibodies that specifically bind to MSLN or cells that produce antibody that specifically binds to MSLN;

step (a) is performed before or during the period in which the animal is establishing central tolerance.

In some preferred embodiments, the animal is a rodent, optionally a mouse.

In some preferred embodiments:

(i) step (a) is performed before or during the eight days after birth of the animal, before or during the ten days after birth of the animal or for the lifetime of the animal, and/or

(ii) step (b) is performed later than eight days after birth or later than ten days after birth.

In some preferred embodiments:

(i) step (a) comprises inducing inactivation of endogenous MSLN expression, and/or

(ii) step (b) comprises inducing expression of human MSLN in the animal.

In some preferred embodiments, the animal comprises:

(i) one or more genes encoding human MSLN and

(ii) one or more genes that encode:

(1) a transactivator that enhances expression of human MSLN, and/or

(2) an inducible nuclease that enhances expression of human MSLN.

In some preferred embodiments, the inducible nuclease is Cre recombinase (Cre) , flippase (Flp) , a CRISPR associated protein (Cas) or a nuclease that does not cut the DNA of the animal.

In some preferred embodiments:

(i) the one or more genes encoding human MSLN are transgenes, and/or

(ii) the one or more genes encoding the transactivator and/or inducible nuclease are transgenes.

In some preferred embodiments, the transactivator is reverse tetracycline-controlled transactivator (rtTA) , and wherein a Tet Response Element (TRE) is upstream of the gene encoding human MSLN.

In some preferred embodiments, the animal expresses heavy chain-only antibodies.

In some preferred embodiments, the animal expresses tetrameric antibodies comprising two heavy and two light chains.

In some preferred embodiments, the animal comprises one or more transgenic immunoglobulin locus.

In some preferred embodiments, the one or more transgenic immunoglobulin loci comprise:

(i) one or more heterologous heavy chain loci, each of which comprises at least one V gene segment, at least one D gene segment, at least one J gene segment and at least one constant region; and/or

(ii) one or more heterologous light chain loci, each of which comprises at least one V gene segment, at least one J gene segment and at least one constant region.

In some preferred embodiments:

(i) one or more endogenous immunoglobulin heavy chain loci in the animal are deleted or silenced and/or

(ii) one or more endogenous immunoglobulin light chain loci in the animal are deleted or silenced.

In the third aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof of the invention.

In the fourth aspect, the invention provides vector comprising the nucleic acid of the invention.

In the fifth aspect, the invention provides a host cell comprising the nucleic acid or the vector of the invention.

In the sixth aspect, the invention provides a pharmaceutical composition comprising (i) the antibody or the antigen binding fragment thereof of the invention; and (ii) a pharmaceutically acceptable carrier or excipient.

In the seventh aspect, the invention provides an antibody-drug conjugate (ADC) , comprising the antibody or the antigen binding fragment thereof of the invention.

In the eighth aspect, the invention provides a method of treating a cancer in a subject, comprising administering to the subject an effective amount of the antibody or the antigen binding fragment thereof, the pharmaceutical composition, or the ADC of the invention. In some preferred embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer and ovarian cancer.

In some preferred embodiments, the method further comprises administering to the subject a second therapeutic agent. Preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.

In a ninth aspect, the invention provides an effective amount of the antibody or the antigen binding fragment thereof, the pharmaceutical composition, or the ADC of the invention for use in a method of treating a cancer in a subject.

In a tenth aspect, the invention provides the use of the antibody or the antigen binding fragment thereof, the pharmaceutical composition, or the ADC of the invention in the manufacture of a medicament for treating a cancer in a subject.

In an eleventh aspect, the invention provides a method for diagnosing mesothelin-positive cancer in a subject comprising:

(a) obtaining a biological sample from the subject,

(b) contacting the sample with the antibody or the antigen binding fragment thereof of the invention, and

(c) detecting binding of the antibody to the sample,

wherein an increase in binding of the antibody or antigen binding fragment thereof to the sample as compared to binding of the antibody or antigen binging fragment thereof to a control sample identifies the subject as having a mesothelin-positive cancer.

In a twelfth aspect, the invention provides a method for imaging a mesothelin-positive cancer in a subject comprising:

(a) administering the antibody or antigen binding fragment thereof of the invention to the subject, wherein the antibody is conjugated to a detectable marker, and

(b) detecting the presence of the marker.

In some preferred embodiments, (a) the detectable marker is ¹¹¹In, and preferably the detection of the marker is by single-photon emission computed tomography, or

(b) the detectable marker is ⁸⁹Zr, and preferably the detection of the marker is by positron emission tomography.

In a thirteenth aspect, the invention provides a method for producing an antigen-specific antibody in a transgenic non-human animal comprising:

(A) inducing expression of the antigen in the animal, and

In some preferred embodiments, the method further comprises:

(A’) reducing or eliminating expression of a protein that is similar or identical to the antigen in the animal,

In some preferred embodiments, the animal is a rodent, optionally a mouse.

In some preferred embodiments:

(ii) step (A) is performed later than eight days after birth.

In a fourteenth aspect, the invention provides a method for producing an antigen-specific antibody in a non-human animal comprising:

(b) immunising the animal with the antigen, and

In some preferred embodiments, the animal is a rodent, optionally a mouse.

In some preferred embodiments:

(i) step (a) comprises inducing inactivation of protein expression, and/or

(ii) step (b) comprises inducing expression of the antigen in the animal.

In some preferred embodiments, the animal comprises:

(i) one or more genes encoding the antigen and

(ii) one or more genes that encode:

(1) a transactivator that enhances expression of the antigen, and/or

(2) an inducible nuclease that enhances expression of the antigen.

In some preferred embodiments:

(i) the one or more genes encoding the antigen are transgenes, and/or

In some preferred embodiments, the transactivator is reverse tetracycline-controlled transactivator (rtTA) , and wherein a Tet Response Element (TRE) is upstream of the gene encoding the antigen.

In some preferred embodiments, the antigen is:

(i) a protein complex, optionally wherein the antigen is encoded by more than one gene, or

(ii) a posttranslational modification in a protein complex, such as a sugar moiety.

In some preferred embodiments:

(i) the protein is an endogenous protein, and/or

(ii) the amino acid sequence of the protein is at least 90%, at least 95%, at least 99%or 100%identical to the amino acid sequence of the antigen.

In some preferred embodiments:

(i) the protein is in a first form, and

(ii) expression of the protein in a second form is not reduced or eliminated.

In some preferred embodiments, the first and second forms are splice variants.

In some preferred embodiments:

(i) the first form is a membrane-bound form, the second form is a secreted form and the antigen is a protein in membrane-bound form, or

(ii) the first form is a secreted form, the second form is a membrane-bound form and the antigen is a protein in secreted form.

In some preferred embodiments:

(i) the protein is an endogenous protein that is essential for development of the animal, and

(ii) the method further comprises introducing a gene that encodes a replacement protein that has a similar function to the endogenous protein but is sufficiently different in amino acid sequence to the antigen to allow immunisation.

In some preferred embodiments:

(i) the amino acid sequence of the replacement protein is less than 80%identical, less than 70%identical, less than 60%identical or less than 50%identical to the amino acid sequence of the antigen, and/or

(ii) the replacement protein is obtained from an animal of a different species.

In some preferred embodiments, the gene encoding the replacement protein replaces a gene encoding the endogenous protein.

In some preferred embodiments:

In a fifteenth aspect, the invention further provides a method of producing an antigen-specific antibody in a non-human animal comprising:

(b) immunising the animal with the antigen, and

In a sixteenth aspect, the invention provides an antigen-specific antibody or an antigen binding fragment thereof obtained by any of the method according to the thirteenth aspect, the fourteenth aspect or the fifteenth aspect of the invention.

In some preferred embodiments of the sixteenth aspect, wherein the antibody is an antibody that specifically binds to mesothelin.

DESCRIPTION OF THE DRAWINGS

Figure 1. Scheme of generating anti-mesothelin H2L2 antibodies. Harbour Mice H2L2 transgenic mice are immunized by repeated injection with recombinant mesothelin protein, followed by hybridoma generation and clone screening to identify MSLN-specific tetrameric (H2L2) antibodies.

Figure 2. Scheme of generating of anti-mesothelin HCAb antibodies. Harbour Mice HCAb transgenic mice are immunized by induction of human mesothelin expression via transgenesis in the mice post weaning, followed by HCAb cDNA library generation and screening to identify MSLN-specific heavy chain-only antibodies (HCAb) .

Figure 3. Exemplary schemes for generating mice that are capable of inducible expression of an antigen-encoding gene (areceiver gene) . In these schemes, expression of the antigen is induced after the tolerance period, thereby eliciting an antibody response against the antigen.

Figure 4. Exemplary schemes for replacing an endogenous gene that encodes a similar or identical protein to the antigen of interest with a gene that encodes a functionally similar protein that has a sufficiently different structure from the antigen of interest, thereby avoiding tolerance being established for the antigen of interest, without eliminating the activity associated with the endogenous protein, which may be important for development.

Figure 5. Exemplary scheme for engineering the expression of a secreted form of a protein antigen during the mouse tolerance period, followed by induced expression of the membrane-bound form of a protein antigen after the mouse tolerance period, resulting in the generation of antibodies that are specific for the membrane-bound form. The Cre-lox system is shown as an exemplary means for achieving the selective expression of the membrane-bound form after the tolerance period.

Figure 6. pTRE-Tight Vector information. pTRE-Tight Vector contains an MCS immediately downstream of the Tet-responsive Ptight promoter. cDNAs or genes inserted into the MCS will be responsive to the tTA and rtTA regulatory proteins in the Tet-Off and Tet-On systems, respectively. Ptight contains a modified Tet response element (TREmod) , which consists of seven direct repeats of a 36-bp sequence that contains the 19-bp tet operator sequence (tetO) . The TREmod is just upstream of the minimal CMV promoter (PminCMVΔ) , which lacks the enhancer that is part of the complete CMV promoter. Consequently, Ptight is silent in the absence of binding of TetR or rTetR to the tetO sequences. In some cases, addition of a Kozak consensus ribosome binding site may improve expression levels; however, many cDNAs have been efficiently expressed in Tet systems without the addition of a Kozak sequence. pTRE-Tight-Gene X plasmids should be co-transfected with the Linear Hygromycin Marker (such as ClonTech Cat. No. 631625) or Linear Puromycin Marker (such as ClonTech Cat. No. 631626) to permit selection of stable transfectants. pTRE-Tight was derived from pTRE, originally described as pUHD10-3 in Resnitzky (1994) Mol. Cell. Biol. 14: 1669-1679.

Figure 7. The process and results of the doxycycline-induced immunisation of H2L2 mice against Her2. Panel A depicts the process for the generation of the stock line of mice that contain the H2L2 locus and ubiquitously express the rtTA transactivator protein from mice that have been generated previously (WO 2014/141189 and Katsantoni et al 2007) . Panel B depicts the process for the generation of the H2L2 mice containing both the rtTA and TRE-HER2 transgene using microinjection of the HER2 gene in fertilised eggs. Immunisation is started by doxycycline added to the drinking water. Panel C depicts the results of detection of the antibodies in the mice. The left panel shows an example of an ELISA assay in which HER2 protein is fixed in plastic wells, serum is added and a colour change is detected for the presence of bound antibody. A dilution series (1/10, 1/100, and 1/500) of the serum in this assay is shown. As shown in the right panel, 5 (designated as 226, 227, 340, 18-415, and 208) out of 8 of the H2L2 mice on doxycycline showed a specific response to HER2.

Figure 8. The process and results of the doxycycline-induced immunisation of HCAb mice (8V3 and 9V3) against Her2. Panel A depicts the process for the generation of the stock line of mice that contain the HCAb locus and ubiquitously express the rtTA transactivator protein from mice that have been generated previously (WO 2014/141189 and Katsantoni et al 2007) . Panel B depicts the process for the generation of the HCAb mice containing both the rtTA and TRE-HER2 transgene using microinjection of the HER2 gene in fertilised eggs. Immunisation is started by doxycycline added to the drinking water. Panel C depicts the results of detection of the antibodies produced in the mice. The panels show an example of results from an ELISA assay in which HER2 protein is fixed in plastic wells, and a dilution series of serum is added and a colour change is detected for the presence of bound antibody. 4 (designated as 188, 18-408, 18-411, and 18-413) out of 7 of the 8V3 mice and 5 (i.e. 18-406, 18-407, 18-416, 18-417, and 18-420) out of 5 of the 9V3 mice on doxycycline showed a specific response to HER2.

Figure 9. Doxycycline-induced immunisation of HCAb mice (9V3) against mesothelin. 9V3 HCAb mice were generated identical to those shown in Figure 8 but carried an inducible mesothelin gene rather than HER2. The dilution series on the serum of 2 out of 7 mice showed a positive response to human mesothelin. Further analysis showed that some antibodies specifically recognize the membrane bound form of mesothelin.

Figure 10. The work flow of screening strategy and process for Single B-cell Cloning Screening.

Figure 11. Binding of H2L2 antibodies to membrane-bound human MSLN on the surface of CHOK1-human MSLN cells.

Figure 12. Binding of H2L2 antibodies to membrane-bound cynomolgus MSLN on the surface of CHOK1-cyno MSLN cells.

Figure 13. Binding of H2L2 antibodies to membrane-bound human MSLN endogenously expressed on COV644 cell line.

Figure 14. Binding of HCAb antibodies to MSLN-expressing cells: (A) CHOK1-human MSLN cells; (B) CHOK1-cyno MSLN cells; (C) COV644 cells.

Figure 15. Binding of HCAb antibodies to MSLN-expressing cells: (A) CHOK1-human MSLN cells; (B) CHOK1-cyno MSLN cells; (C) COV644 cells.

Figure 16. Epitope binning matrix of H2L2 antibodies. A rate >50%indicates the two antibodies binds to different epitope bins; a rate <20%indicates an overlapping bin of two assessed antibodies.

Figure 17. Internalization of antibodies on COV644 cells.

Figure 18. Binding of H2L2 antibodies (PR300147, PR300281, PR300163, PR300283) to COV644 cells in the presence or absence of soluble MSLN (sMSLN) : (A) without sMSLN; (B) with 90 nM sMSLN.

Figure 19. Binding of H2L2 antibodies (PR300162, PR300284) to COV644 cells in the presence or absence of soluble MSLN (sMSLN) : (A) without sMSLN; (B) with 90 nM sMSLN.

Figure 20. Binding of H2L2 antibodies (PR300187, PR300193, PR300286) to COV644 cells in the presence or absence of soluble MSLN (sMSLN) : (A) without sMSLN; (B) with 90 nM sMSLN.

Figure 21. Binding of HCAb antibodies to COV644 cells in the presence (white bar) or absence (black bar) of soluble MSLN. 250,000 cells were loaded per well with or without 1μg his-tagged soluble MSLN. The controls were amatuximab group (at 1μg) and the secondary antibody alone (C-+2nd group) .

Figure 22. Binding of HCAb antibodies to COV644 cells in the absence (-sMSLN; dashed line) or presence (+sMSLN; solid line) of 60 nM soluble MSLN, as determined by FACS.

Figure 23. HCAb 51 8-10 binds specifically to the membrane bound form of mesothelin. The figure shows binding of HCAb 51 8-10 and amatuximab to biotin-MSLN bound to streptavidin tips (Octet) in the presence of 60 nM soluble MSLN.

Figure 24. H2L2 and HCAb antibodies block the interaction between MSLN and MUC16 on HeLa cells, as determined by FACS. HeLa cells were mixed with 0.2μg of biotin-tagged soluble MSLN with either 0.5μg HCAb (black bars) or 1μg of H2L2 (white bars) . Positive binding was determined using Strep-PE. No antibody was added in the positive control group (C+) , no MSLN was added in the C-2nd group (Strep-PE only control) , and neither antibody nor MSLN was added in the C-control group (HeLa cells only) . “141” and “11” on this figure represent 141pl1-4 and 11pl1-4 clones respectively.

Figure 25. HCAb antibodies block the interaction between MSLN and MUC16 on HeLa cells, as determined by FACS. HeLa cells were mixed with 0.25μg biotinylated MSLN in the presence of 1μg of HCAb. Positive binding was determined using Strep-PE. 2μg of amatuximab was used as a positive control (C+Amatuximab) and an irrelevant antibody was used as a negative control (C-antibody) . The C-2nd group with no antibody and MSLN added was used to measure the background of the Strep-PE signal.

Figure 26. HCAb antibodies block the MSLN-MUC16 interaction by BLI. Biotinylated MSLN was loaded onto the Octet tips followed by binding of the antibodies. The extracellular domain of MUC16 was added (point 0 in the graph) subsequently, and its binding was recorded over time followed by a washing step, which indicates the stability of the interaction. Top dashed line in the graph is an isotype control.

Figure 27. ADCC assay with anti-MSLN antibodies on CHO cells expressing human MSLN.

Figure 28. HCAb 11A10 (PR004197) and its derived variants (PR006372, PR006373) bind to MSLN-expressing cells: (A) COV644, (B) CHOK1-cyno MSLN.

SEQUENCE LISTING

The sequences of the light chain, heavy chain, the variable region of light chain, the variable region of heavy chain, the CDRs of the light chain and heavy chain are indicated in Tables 1-6 below.

Table 1. Sequences of the heavy chain and light chain of H2L2 antibodies

Table 2. Sequences of the heavy chain variable region and light chain variable region of H2L2 antibodies

Table 3. Sequences of the HCDR1-3 and LCDR1-3 of H2L2 antibodies (EU Kabat system)

Table 4. Sequences of the heavy chain of HCAb antibodies

Table 5. Sequences of the heavy chain variable region of HCAb antibodies

Table 6. Sequences of the heavy chain CDR1-3 of HCAb antibodies (EU Kabat System)

In one aspect, the invention provides regular heavy and light chain antibodies (H2L2) and heavy chain only antibodies (HCAb) that either bind the soluble form of mesothelin or only bind the membrane bound form of MSLN for diagnostic and therapeutic purposes to detect and image these tumours to allow more precise surgery and diagnosis and provide a novel therapeutic agent for this lethal disease. Such antibodies would provide a realistic chance of longer-term survival or cure for patients suffering from MSLN expressing tumors.

Accordingly, antibodies that bind specifically to membrane bound mesothelin may be much better suited to diagnosis/imaging of the primary tumour and detection of metastasis to allow accurate surgery of a tumour and for radiotherapy or immunotherapy, because much lower doses of antibodies could be used particularly in combination with antibodies that would prevent the interaction of mesothelin with MUC16.

DETAILED DESCRIPTION OF THE INVENTION

The aforementioned features and advantages of the invention as well as additional features and advantages thereof will be more clearly understood hereafter as a result of a detailed description of the following embodiments when taken in conjunction with the drawings.

The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present invention. The embodiments shall not be construed to limit the scope of the present invention. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations) " , Helvetica Chimica Acta (1995) , CH-4010 Basel, Switzerland; Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed. ) , Vols. 1-3, Cold Spring Harbor Laboratory Press (1989) ; F. Ausubel et al, eds., "Current protocols in molecular biology" , Green Publishing and Wiley InterScience, New York (1987) ; Roitt et al., "Immunology (6th Ed. ) , Mosby/Elsevier, Edinburgh (2001) ; and Janeway et al., "Immunobiology" (6th Ed. ) , Garland Science Publishing/Churchill Livingstone, New York (2005) , as well as the general background art cited above.

As used herein, singular forms “a” , “and, ” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.

Unless indicated or defined otherwise, the term "comprise" , and variations such as "comprises" and "comprising" , should be understood to imply the inclusion of a stated elements or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The term “about” in relation to a numerical value x is optional and means, for example, x+10%.

Antibodies

In some embodiments, the antibody of the invention is a polyclonal, monoclonal, multispecific, mouse, human, humanized, primatized or chimeric antibody or a single-chain antibody. The term “antibody” encompasses entire tetrameric antibodies and antigen-binding fragments thereof. In some embodiments, the antigen-binding fragment thereof is selected from a V_H domain, Fab, Fab', F (ab') 2, Fd, Fv, a single-chain Fv (scFv) and a disulfide-linked Fv (sdFv) . The term “antibody” also encompasses each of the following: scFv-Fc, diabody, scFv-CH3 (minibody) , scFab, scFv-zipper, tandem Fc and IgG hexamer.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V_H domain associated with a V_L domain, the V_H and V_L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V_H -V_H, V_H -V_L or V_L -V_L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V_H or V_L domain.

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V_H -C_H1 ; (ii) V_H -C_H2; (iii) V_H -C_H3; (iv) V_H -C_H1 –C_H2; (V) V_H –C_H1 –C_H2-C_H3; (vi) V_H -C_H2-C_H3; (vii) V_H -C_L; (viii) V_L -C_H1 ; (ix) V_L -C_H2; (x) V_L -C_H3; (xi) V_L -C_H1 -C_H2; (xii) V_L -C_H1 -C_H2-C_H3; (xiii) V_L -C_H2-C_H3; and (xiv) V_L -C_L.

In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge or linker region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V_H or V_L domain (e.g., by disulfide bond (s) ) .

As with full antibody molecules, antigen-binding fragments may be mono-specific or multi-specific (e.g., bi-specific) . A multi-specific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antibody format, including the exemplary bi-specific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

In some embodiments, scFv are fused to constant immunoglobulin domains. For example, in some embodiment, scFv are fused to constant immunoglobulin domains of tetrameric antibodies (e.g. Ig-scFv) , scFv-Fc (e.g. scFv₂-Fc) , F (ab’) ₂ (e.g. F (ab’) ₂-scFv₂) , Fab (e.g. Fab-scFv) or heavy chain-only antibodies.

In some embodiments, the antibody is tetravalent (and optionally bispecific) , such as taFv-Fc, scDb-Fc, scDb-CH3, Db-Fc, scFv₂-H/L chain.

In some embodiments, scFv or Fab are fused to homo-or hetero-dimerizing peptides (e.g. scFv-Jun/Fos, Fab’-Jun/Fos or scFv-dhlx-scFv) .

In some embodiments, the antibody contains an Fc domain or a portion thereof that binds to the FcRn receptor. As a non-limiting example, a suitable Fc domain may be derived from an immunoglobulin subclass such as IgA, IgE, IgG or IgM. In some embodiments, a suitable Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. Particularly suitable Fc domains include those derived from human antibodies.

In some embodiments, the antibody is a tetrameric bivalent antibody that consists of two heavy chains (either V_H, C_H1, C_H2 and C_H3 or V_H, C_H1, C_H2, C_H3 and C_H4) and two light chains (V_L and C_L) . In some embodiments, the antibody is conjugated to a fluorescent label or cytotoxic agent. In some embodiments, the antibody is a heavy chain-only antibody that consists of two heavy chains (either V_H, C_H1, C_H2 and C_H3 or V_H, C_H1, C_H2, C_H3 and C_H4) . In some embodiments, the antibody is conjugated to a fluorescent label or cytotoxic agent.

In some embodiments, the antibody consists of a first heavy chain (V_H, C_H2 and C_H3 or V_H, C_H2, C_H3 and C_H4) and a second heavy chain (scFv, C_H2 and C_H3 or scFv, C_H2, C_H3 and C_H4) .

In some embodiments, the antibody consists of a first heavy chain (scFv, V_H, CH₂ and C_H3) and a second heavy chain (scFv, V_H, CH₂ and C_H3) .

In some embodiments, the antibody consists of a first heavy chain (scFv, V_H, CH₂, C_H3 and C_H4) and a second heavy chain (scFv, V_H, CH₂, C_H3 and C_H4) .

In some embodiments, the antibody consists of a first heavy chain (V_H, scFv, CH₂ and C_H3) and a second heavy chain (V_H, scFv, CH₂ and C_H3) .

In some embodiments, the antibody consists of a first heavy chain (V_H, scFv, CH₂, C_H3 and C_H4) and a second heavy chain (V_H, scFv, CH₂, C_H3 and C_H4) .

In some embodiments, the antibody consists of a first heavy chain (scFv, CH₂, C_H3 and V_H) and a second heavy chain (scFv, CH₂, C_H3 and V_H) .

In some embodiments, the antibody consists of a first heavy chain (scFv, CH₂, C_H3, C_H4 and V_H) and a second heavy chain (scFv, CH₂, C_H3, C_H4 and V_H) .

In some embodiments, the antibody consists of a first heavy chain (V_H, CH₂, C_H3 and scFv) and a second heavy chain (V_H, CH₂, C_H3 and scFv) .

In some embodiments, the antibody consists of a first heavy chain (V_H, CH₂, C_H3, C_H4 and scFv) and a second heavy chain (V_H, CH₂, C_H3, C_H4 and scFv) .

In some embodiments, the antibody is a human antibody.

The invention further provides an antigen-binding fragment of the invention (e.g. a V_H domain, a V_L domain, a Fab, a F (ab’) ₂, a scFv, a sc (Fv) ₂, or a diabody) linked to a constant region comprising one or more constant domains (e.g. C_H1, C_H1-C_H2, C_H1-C_H2-C_H3, C_H1-C_H2-C_H3-C_H4, C_H2-C_H3, or C_H2-C_H3-C_H4) . In some embodiments, one or more antigen-binding fragments (e.g. V_H domains) is linked to the N-terminus of the constant region. In some embodiments, one or more antigen-binding fragments (e.g. V_H domains) is linked to the C-terminus of the constant region. In some embodiments, one or more antigen-binding fragments (e.g. V_H domains) is linked to the N-terminus and one or more antigen-binding fragments (e.g. V_H domains) is linked to the C-terminus of the constant region.

Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest, " National Institutes of Health, Bethesda, Md. (1991) ; Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997) ; and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989) . Public databases are also available for identifying CDR sequences within an antibody.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific comprising a first binding specificity to a first epitope in the MSLN protein and a second binding specificity to a second epitope in the MSLN protein wherein the first and second epitopes are distinct and non-overlapping.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are tri-specific comprising a first binding specificity to a first epitope in the MSLN protein, a second binding specificity to a second epitope in the receptor binding domain of MSLN protein and a third binding specificity to a third epitope in the MSLN protein, wherein the first, second and third epitopes are distinct and non-overlapping.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are quadri-specific comprising a first binding specificity to a first epitope in the MSLN protein, a second binding specificity to a second epitope in the MSLN protein, a third binding specificity to a third epitope in the MSLN protein and a fourth binding specificity to a fourth epitope in the MSLN protein, wherein the first, second, third and fourth epitopes are distinct and non-overlapping.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are multispecific comprising multiple binding specificities for epitopes in the MSLN protein that are distinct and non-overlapping.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific comprising a first binding specificity to a first epitope in the MSLN protein and a second binding specificity to a second epitope in the MSLN protein, wherein the first binding specificity is for membrane bound MSLN, and wherein binding to the second epitope blocks the interaction between MSLN and MUC16.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific comprising a first variable region and a second variable region described herein. In some embodiments, the first variable region specifically binds to membrane bound MSLN and the second variable region specifically binds to MSLN and blocks the interaction between MSLN and MUC16.

Conjugated antibodies

In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody) . The effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, carbohydrate or recombinant virus. An antibody conjugate is often referred to as an "immunoconjugate. " When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent) , the conjugate is often referred to as an "antibody-drug conjugate" or "ADC. " Other antibody conjugates include, for example, multi-specific (such as bispecific or trispecific) antibodies.

In some embodiments, the effector molecule can be a detectable label or an immunotoxin. Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40) , diphtheria toxin (DT) , botulinum toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as the domain la of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody. The term "conjugated" or "linked" may refer to making two polypeptides into one contiguous polypeptide molecule. In one embodiment, an antibody is joined to an effector molecule. In another embodiment, an antibody joined to an effector molecule is further joined to a lipid or other molecule to a protein or peptide to increase its half-life in the body. The linkage can be either by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule.

The invention provides immunoconjugates that include a monoclonal antibody or antigen-binding fragment disclosed herein and an effector molecule. In some embodiments, the effector molecule is a toxin, such as, but not limited to, Pseudomonas exotoxin or a variant thereof. In other embodiments, the effector molecule is a detectable label, such as, but not limited to, a fluorophore, an enzyme or a radioisotope.

The disclosed monoclonal antibodies can be conjugated to a therapeutic agent or effector molecule. Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody. A therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule. One of skill in the art will appreciate that therapeutic agents can include various drugs such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) that contain pharmacological compositions, radioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and other labels, target moieties and ligands.

The choice of a particular therapeutic agent depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the therapeutic agent can be a cytotoxin that is used to bring about the death of a particular target cell (such as a tumor cell) . Conversely, where it is desired to invoke a non-lethal biological response, the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.

With the therapeutic agents and antibodies described herein, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same effector moiety or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

Effector molecules can be linked to an antibody of interest using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH) , free amine (-NH₂) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector molecule from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site.

Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules) , drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein) . In general, the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody) , a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a strep tavidin core region or a polyhistidine tag) .

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies) . Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate) . Such linkers are commercially available.

The antibody can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT) , computed axial tomography (CAT) scans, magnetic resonance imaging (MRI) , nuclear magnetic resonance imaging NMRI) , magnetic resonance tomography (MTR) , ultrasound, fiberoptic examination, and laparoscopic examination) . Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI) . For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) . An antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

An antibody may be fused to a self-labelling protein tag (e.g. HaloTag) . For example, the protein tag could be cloned at the end of a constant region. HaloTag is a self-labelling protein tag derived from a bacterial enzyme (ahaloalkane dehalogenase) , designed to covalently bind to a synthetic ligand. In some instances, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3 (6) : 373-82) . An antibody may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) , and manganese.

Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. An antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) . In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

An antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG) , a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.

Toxins can be employed with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO) . Contemplated toxins also include variants of the toxins described herein (see, for example, see, U.S. Patent Nos. 5,079,163 and 4,689,401) . In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095) . As used herein "Pseudomonas exotoxin" refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al. Biol. Chem. 264: 14256-14261, 1989) .

Provided herein are ADCs that include a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (such as specifically binds) mesothelin. In some embodiments, the drug is a small molecule. In some examples, the drug is a cross-linking agent, an anti-microtubule agent and/or anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells.

Exemplary cytotoxic agents include, but are not limited to, a PDB, an auristatin, a maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracycline, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, a combretastain, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a puromycin, a tubulysin, a hemiasterlin, a spliceostatin, or a pladienolide, as well as stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD) . The natural product anthramycin (a PBD) was first reported in 1965 (Leimgruber et al, J Am Chem Soc, 87: 5793-5795, 1965; Leimgruber et al., JAm Chem Soc, 87: 5791-5793, 1965) . Since then, a number of PBDs, both naturally-occurring and synthetic analogues, have been reported (Gerratana, Med Res Rev 32 (2) : 254-293, 2012; and U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099) . As one example, PDB dimers recognize and bind to specific DNA sequences, and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties (see, for example, US 2010/0203007) . Exemplary linkage sites on the PBD dimer include the five-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (see WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; and WO 2011/130598) .

In some embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, and are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111) . Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042) . Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4, 308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

In some embodiments, the ADC includes an antibody conjugated to a dolastatin or auristatin, or an analog or derivative thereof (see U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431) . Auristatins are derivatives of the marine mollusk compound dolastatin-10. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al., Antimicrob Agents and Chemother 45 (12) : 3580-3584, 2001) and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42: 2961-2965, 1998) . Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB) , auristatin EFP (AEFP) , MM AD (Monomethyl Auristatin D or monomethyl dolastatin 10) , MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine) , MMAE (Monomethyl Auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine) , 5-benzoylvaleric acid-AE ester (AEVB) , and other auristatins (see, for example, U.S. Publication No. 2013/0129753) .

In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics, and analogues thereof, are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al, Cancer Res 53: 3336-3342, 1993; Lode et al, Cancer Res 58: 2925-2928, 1998) . Exemplary methods for preparing ADCs with a calicheamicin drug moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

In some embodiments, the ADC comprises an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. It is believed that anthracyclines can operate to kill cells by a number of different mechanisms, including intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis; inducing production of free radicals which then react with cellular macromolecules to cause damage to the cells; and/or interactions of the drug molecules with the cell membrane. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin and mitoxantrone, and derivatives thereof. For example, PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al, Clin Cancer Res 11 (4) : 1608-1617, 2005) . Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin (Grandi et al, Cancer Treat Rev 17: 133, 1990; Ripamonti et al, Br J Cancer 65: 703-707, 1992) .

In some embodiments, the ADC can further include a linker. In some examples, the linker is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties to an antibody to form an ADC. In some embodiments, ADCs are prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, a cysteine thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.

In some examples, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Exemplary linkers with such reactive functionalities include maleimide, haloacetamides, oc-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

In some examples, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some cases, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Non-limiting examples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.

In some examples, the linker is a cleavable linker, which facilitates release of the drug. Examples of cleavable linkers include acid-labile linkers (for example, comprising hydrazone) , protease-sensitive linkers (for example, peptidase-sensitive) , photolabile linkers, and disulfide-containing linkers (Chari et al, Cancer Res 52: 127-131, 1992; U.S. Patent No. 5,208,020) .

The ADCs disclosed herein can be used for the treatment of a mesothelin-positive cancer alone or in combination with another therapeutic agent and/or in combination with any standard therapy for the treatment of cancer (such as surgical resection of the tumor, chemotherapy or radiation therapy) .

Antibody-conjugated nanoparticles

In an embodiment, the nanoparticle of the antibody-conjugated nanoparticle has a diameter of between 1 and 500 nm. Preferably, the nanoparticle has a diameter of between 150 and 400 nm, such as 200 to 400 nm. The diameter of a nanoparticle may be measured by any suitable method, such as dynamic light scattering, nanoparticle tracking analysis, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, photo correlation spectroscopy, x-ray diffraction, or time of flight mass spectroscopy. Preferably, the diameter of a nanoparticle is measured by dynamic light scattering or nanoparticle tracking analysis. The diameter of the nanoparticle is preferably measured when the nanoparticle comprises the payload, most preferably when the nanoparticle has encapsulated the payload such that the payload is in the interior of the nanoparticle.

Preferably, the nanoparticles are biodegradable. Additionally, the nanoparticles are preferably non-toxic, more preferably non-toxic to a human patient.

In an embodiment, the nanoparticles have a negative zeta potential. In an embodiment, the zeta potential is between -30 and 0 mV, such as between -25 and -5 mV.

In an embodiment, the nanoparticle comprises one or more of: chitosan, alginate, xanthan gum, cellulose, poly (lactic-co-glycolic acid) , polyethylene glycol, poly (propylene glycol) , poly (aspartic acid) , poly (lactic acid) . In an embodiment, the nanoparticle is one of: a liposome, a polymeric micelle, a dendrimer.

In an embodiment, the nanoparticle comprises biodegradable polymers.

The nanoparticle may be surface modified, for example a nanoparticle may be coated with polyethylene glycol (PEG) .

The nanoparticle may be synthesised using any suitable method, for example solvent extraction, microfluidic nanoparticle production, dialysis, solution casting, polycarbonate membrane extrusion, high pressure homogenisation, reversed phase evaporation, sonication, or lipid film hydration sonication extrusion.

In an embodiment, the nanoparticle comprises poly (lactic-co-glycolic acid) (PLGA) . In an embodiment, the nanoparticle comprises polyethylene glycol (PEG) . In a preferred embodiment, the nanoparticle comprises PLGA and PEG. More preferably, the nanoparticle consists essentially of PLGA and PEG, and optionally the nanoparticle consists of PLGA and PEG. In an embodiment, the nanoparticle consists of a PLGA core that is surface coated in PEG.

A nanoparticle comprising or consisting of PLGA and/or PEG may be synthesised by any suitable method, such as emulsification-evaporation, salting out, nanoprecipitation, or using microfluidics (see, e.g., Danhier 2012 Journal of Controlled Release 161 (2) : 505-522) .

The nanoparticle is preferably capable of being taken up by a cell and releases the payload inside the cell, thereby delivering the payload directly to the intracellular space. In an embodiment, the antibody-conjugated nanoparticle is taken up by a cell by pinocytosis. Particularly when the nanoparticle has a diameter less than 500 nm, the nanoparticle is taken up by a cell by pinocytosis.

In an embodiment, the antibody-conjugated nanoparticle is taken up by a cell by phagocytosis. In an embodiment, the nanoparticle is configured to release the payload in a pH-dependent manner, for example the payload is released when the pH is below 7, 6.5, 6, 5.5, or 5.

In embodiments of the antibody-conjugated nanoparticle, the nanoparticle is preferably conjugated to the antibody at the antibody Fc region, i.e. the antibody constant region.

In an embodiment, the nanoparticle and antibody are conjugated such that the antibody is randomly oriented on the surface of the nanoparticle. In a more preferred embodiment, the nanoparticle and antibody are conjugated such that the antibody is oriented with the antigen binding site facing away from the nanoparticle core, allowing optimal engagement with target molecules.

In an embodiment, the nanoparticle is conjugated to the antibody via an ester bond to an amino group on the antibody. In an embodiment, the amino group is from a lysine residue in the antibody Fc region. In an embodiment, the conjugation is via an NHS-ester reaction.

In an embodiment, the nanoparticle is conjugated to the antibody via a thioether bond to a thiol group on the antibody. In an embodiment, the amino group is from a cysteine residue in the antibody Fc region. In an embodiment, the cysteine residue is a C-terminal cysteine, i.e. the cysteine residue is at the C-terminal of the Fc region. In an embodiment, the cysteine residue is near the C-terminal of the Fc region, such as anywhere within 20 residues of the C-terminal, such as within 15, 10, 5, 4, 3, or 2 residues of the C-terminal. In an embodiment, the conjugation is via a maleimide-thiol reaction.

In an embodiment, the nanoparticle is conjugated to the antibody in a site-specific manner, i.e. the nanoparticle is conjugated to a specific residue of the antibody, for example the C-terminal residue of the Fc region.

In an embodiment, the nanoparticle is conjugated to one or more antibody molecules. In an embodiment, the nanoparticle is conjugated to 2 or more antibody molecules, such 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 or more antibody molecules. In an embodiment, the nanoparticle is conjugated to a plurality of antibody molecules.

In an embodiment, the obtained nanoparticle further comprises an encapsulated payload.

In an embodiment, the obtained nanoparticle further comprises poly (lactic-co-glycolic acid) (PLGA) .

In an embodiment, the nanoparticle is obtained by a method comprising: dissolving PLGA in dichloromethane (DCM) ; adding a payload to the PLGA-DCM mixture and emulsifying, optionally under sonication; adding the emulsified mixture dropwise to an aqueous phase comprising polyvinyl acetate (PVA) and sonicating the solution; and washing and recovering a nanoparticle comprising PLGA and the encapsulated payload.

In an embodiment, the nanoparticle is freeze dried. In an embodiment, the nanoparticle is rehydrated before conjugation to the antibody.

Alternatively or in addition, the nanoparticle may be conjugated to the antibody using click chemistry. For example, the nanoparticle may be conjugated to the antibody using any one or more of the following reactions: copper (I) -catalyzed azide-alkyne cycloaddition; strain-promoted azide-alkyne cycloaddition; strain-promoted alkyne-nitrone cycloaddition; alkene and azide [3+2] cycloaddition; alkene and tetrazine inverse-demand Diels-Alder; alkene and tetrazole photoclick reaction.

In some embodiments, the nanoparticle is conjugated to the antibody by sortase-mediated transpeptidation (see, e.g. Popp et al. Current Protocols in Protein Science 56 (1) : 15.3.1-15.3.9) .

Preferably, the payload is encapsulated by the nanoparticle such that the payload is in the interior of the nanoparticle. In an embodiment, the payload is encapsulated within the interior of a PLGA core of the nanoparticle. Encapsulation within a nanoparticle protects the payload from premature degradation. Additionally or alternatively, the payload may be conjugated to the nanoparticle.

In an embodiment, the payload is a therapeutic payload, which may be any payload, which may be administered to a patient in need thereof, which treats or ameliorates the symptoms of one or more diseases. In an embodiment, the payload is a medicament, such as a medicament for cancer therapy, for example a chemotherapy drug. In an embodiment, the payload is a toxin, such as an alkylating agent. A toxin as used herein is any molecule which causes the cell to which the payload is delivered to die.

In an embodiment, the payload, optionally a therapeutic payload, comprises at least one of the following: a drug, a protein, RNA, DNA, an imaging agent.

In an embodiment, the payload is a gene editing payload configured to cause double strand breaks in one or more genes or gene loci which are essential for cell survival, such as any gene or gene locus in Wang et al. (2015) Science 350 (6264) : 1096-101. For example, a gene encoding a polymerase or polymerase subunit.

Accordingly, the invention further provides an anti-MSLN antibody according to the invention conjugated to a nanoparticle comprising a therapeutic payload (e.g. a gene editing payload) . In some embodiments, the anti-mesothelin antibody conjugated nanoparticle is for use in treating mesothelin-positive cancer (e.g. mesothelioma, (non-small cell) lung cancer, ovarian cancer or pancreatic cancer) . Accordingly, in one embodiment, an antibody-conjugated nanoparticle comprising a cytotoxic payload, wherein the antibody binds to the membrane-bound form of mesothelin is provided, optionally for use in treating a MSLN-positive cancer (e.g. pancreatic cancer) .

Nucleic acids

The term “polynucleotide” or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

The invention provides nucleic acids encoding anti-MSLN antibodies or portions thereof.

For example, the invention provides nucleic acid molecules encoding any one of the heavy chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the heavy chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the light chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding: (i) any one of the heavy chain variable region sequences disclosed herein and (ii) any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding: (i) any one of the heavy chain variable region sequences disclosed herein and (ii) any one of the light chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding a heavy chain variable region sequence that comprises the CDR sequences of any one of the heavy chain variable region sequences disclosed herein.

In some embodiments, the invention provides nucleic acid molecules encoding a heavy chain variable region sequence that comprises any one of the groups of three CDR sequences disclosed herein.

The invention also provides nucleic acid molecules that encode a heavy chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein.

In some embodiments, the invention provides nucleic acid molecules that encode a heavy chain variable region sequence that comprises CDR1, CDR2 and CDR3 sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR1, CDR2 and CDR3, respectively, of any one of the groups of three CDR sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding a light chain variable region sequence that comprises the CDR sequences of any one of the light chain variable region sequences disclosed herein.

In some embodiments, the invention provides nucleic acid molecules encoding a light chain variable region sequence that comprises any one of the groups of three CDR sequences disclosed herein.

The invention also provides nucleic acid molecules that encode a light chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the light chain variable region sequences disclosed herein.

In some embodiments, the invention provides nucleic acid molecules that encode a light chain variable region sequence that comprises CDR1, CDR2 and CDR3 sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR1, CDR2 and CDR3, respectively, of any one of the groups of three CDR sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding: (i) a heavy chain variable region sequence that comprises the CDR sequences of any one of the heavy chain variable region sequences disclosed herein and (ii) a light chain variable region sequence that comprises the CDR sequences of any one of the light chain variable region sequences disclosed herein. In some embodiments, the invention provides nucleic acid molecules encoding (i) a heavy chain variable region sequence that comprises any one of the groups of three CDR sequences disclosed herein and (ii) a light chain variable region sequence that comprises any one of the groups of three CDR sequences disclosed herein. The invention also provides nucleic acid molecules that encode: (i) a heavy chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein and (ii) a light chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the light chain variable region sequences disclosed herein. In some embodiments, the invention provides nucleic acid molecules that encode (i) a heavy chain variable region sequence that comprises CDR1, CDR2 and CDR3 sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR1, CDR2 and CDR3, respectively, of any one of the groups of three CDR sequences disclosed herein and (ii) a light chain variable region sequence that comprises CDR1, CDR2 and CDR3 sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR1, CDR2 and CDR3, respectively, of any one of the groups of three CDR sequences disclosed herein.

The invention further provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-MSLN antibody. For example, the invention provides recombinant expression vectors comprising any of the nucleic acid molecules mentioned above.

The invention further provides host cells into which any of the vectors mentioned above have been introduced. The invention further provides methods of producing the antibodies and antibody fragments of the invention by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

Pharmaceutical compositions

The invention provides pharmaceutical composition comprising an antibody of the invention. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion) . For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer.

The antibodies or agents of the invention (also referred to herein as “active compounds” ) , and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5%human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation) , transdermal (i.e., topical) , transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA) ; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^TM (BASF, Parsippany, N.J. ) or phosphate buffered saline (PBS) . In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The invention provides therapeutic compositions comprising the anti-MSLN antibodies or antigen-binding fragments thereof of the present invention. Therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM) , DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights) , semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

The dose of antibody may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When an antibody of the present invention is used for treating a disease or disorder in an adult patient, or for preventing such a disease, it is advantageous to administer the antibody of the present invention normally at a single dose of about 0.1 to about 60 mg/kg body weight, more preferably about 5 to about 60, about 10 to about 50, or about 20 to about 50 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, to about 100 mg, or to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Methods of production

1. Inducing expression

In some embodiments, expression of an antigen is induced in a transgenic non-human animal after the animal has established central tolerance.

Immune tolerance is classified into central or peripheral tolerance. If induced in the thymus and bone marrow it is central, when it is induced in other tissues and lymph nodes it is peripheral. The mechanisms by which these forms of tolerance are established are distinct, but the resulting effects are similar. For the purpose of this invention the induction and timing of central tolerance is important. Central tolerance is established by deleting autoreactive lymphocyte clones before they develop into fully immunocompetent cells. In the thymus and bone marrow, maturing lymphocytes are exposed to self-antigens presented by medullary thymic epithelial cells and thymic dendritic cells, or bone marrow cells. The self-antigens correspond to the proteins/peptides that are expressed endogenously. The lymphocytes that express receptors that bind strongly to are removed (deleted) by induction of apoptosis of these autoreactive cells, or by induction of anergy, which is a state of non-activity. Weakly autoreactive B cells may also remain in a state of immunological ignorance and not responding to stimulation of their B cell receptor. Some weakly self-recognizing T cells are alternatively differentiated into regulatory T cells, which act as sentinels in the periphery to calm down potential instances of T cell auto-reactivity. The threshold of deletion is more stringent for T cells than for B cells. It is advantageous for the organism to have B cells recognizing a wide variety of antigen to be able to produce antibodies against a greater diversity of pathogens. However B cells can only be fully activated after confirmation by more self-restricted T cells recognizing the same antigen, thereby keeping autoreactivity (autoimmunity) in check. Thus negative selection ensures that T and B cells will not initiate an immune response to endogenous tissues, while preserving the ability to recognize foreign antigens. Lymphocyte development and education are most active in fetal development and the induction of tolerance is largely finished at 1-2 weeks after birth in the mouse (Wang et al. (2010) J. Immunol. 185: 71-78) . A similar timing for central tolerance establishment is expected for rats. Central tolerance is established in chicken at three weeks after hatching, if not sooner (Yuan F, Li Z. Int Immunol. 2012; 24: 267-72) .

In some embodiments, the methods of the invention comprise immunising a non-human animal with the antigen by inducing expression of the antigen in the animal after the animal has established central tolerance.

In some preferred embodiments, the transgenic non-human animal is a mouse and expression of the antigen is induced in the mouse later than seven days after birth, eight days after birth, later than nine days after birth or later than ten days after birth. In some embodiments, the transgenic non-human animal is a mouse and expression of the antigen is induced in the mouse later than one week, later than two weeks, later than three weeks, later than four weeks, later than five weeks, later than six weeks, later than seven weeks or later than eight weeks after birth.

In some preferred embodiments, the transgenic non-human animal is a rat and expression of the antigen is induced in the rat later than seven days after birth, eight days after birth, later than nine days after birth or later than ten days after birth. In some embodiments, the transgenic non-human animal is a rat and expression of the antigen is induced in the rat later than one week, later than two weeks, later than three weeks, later than four weeks, later than five weeks, later than six weeks, later than seven weeks or later than eight weeks after birth.

In some preferred embodiments, the transgenic non-human animal is a chicken and expression of the antigen is induced in the chicken later than one week, later than two weeks or later than three weeks after hatching.

Any appropriate inducible expression system may be used in the methods of the invention.

In some embodiments, the non-human animal comprises:

(i) one or more genes encoding the antigen; and

(ii) one or more genes that encode:

(1) a transactivator that enhances expression of the antigen, and/or

(2) an inducible nuclease that enhances expression of the antigen.

In some embodiments, the inducible nuclease is Cre recombinase (Cre) , flippase (Flp) or a CRISPR associated protein (Cas) .

In some embodiments, the one or more genes encoding the antigen are transgenes and/or the one or more genes encoding the transactivator and/or inducible nuclease are transgenes.

In some embodiments, a reverse tetracycline-controlled transactivator (rtTA) induction system is used. Advantageously, this is a highly reliable system. In some preferred embodiments, the transactivator is rtTA and a Tet Response Element (TRE) is upstream of the gene encoding the antigen. In some embodiments, doxycycline or a homologue thereof is administered to induce expression of the antigen. In some embodiments, doxycycline is administered to induce expression of the antigen. The doxycycline or homologue thereof may be administered via any appropriate administration route. In some embodiments, the doxycycline or homologue thereof is administered orally (e.g. via drinking water) .

In some embodiments, a tamoxifen-estrogen receptor induction system is used. An exemplary tamoxifen-estrogen receptor induction system is described in Hayashi and McMahon (2002) Developmental Biology 244, 305-318. In some embodiments, tamoxifen or a homologue thereof is administered to induce expression of the antigen. In some embodiments, tamoxifen is administered to induce expression of the antigen. The tamoxifen or homologue thereof may be administered via any appropriate administration route. In some embodiments, the tamoxifen or homologue thereof is administered via injection (e.g. intraperitoneal injection) or orally (e.g. by oral lavage) .

In some embodiments, an inducible nuclease system is used, such as Cre/lox or Flp/frt. For example, a tamoxifen-inducible Cre/lox system is described in Hayashi and McMahon (2002) .

In some embodiments, the transactivator and/or inducible nuclease is introduced directly into non-human animals by homologous recombination, random transgenesis (e.g. oocyte injection) or nuclease-directed integration (e.g. CRISPR or a zinc-finger nuclease) . In some embodiments, the transactivator and/or inducible nuclease is introduced before, during or after a gene encoding the antigen is introduced.

Non-human animals that have immunoglobulin loci and transactivator-and antigen-encoding genes may be produced by:

(1) crossing an existing line of immunoglobulin loci-carrying non-human animals (e.g. the 8V3 HCAb mouse in Example 1) with existing transactivator-encoding gene-carrying animals of the same species (e.g. the transgenic mouse line ubiquitously expressing rtTA2S-M2 described in Katsantoni (2007) ) to generate a stock line of animals that carry a transactivator-encoding gene and immunoglobulin loci, and

(2) introducing an antigen-encoding gene into the stock line of animals.

Non-human animals that have immunoglobulin loci and inducible nuclease-and antigen-encoding genes may be produced by:

(1) crossing an existing line of immunoglobulin loci-carrying non-human animals (e.g. the 8V3 HCAb mouse in Example 1) with existing inducible nuclease-encoding gene-carrying animals of the same species to generate a stock line of animals that carry an inducible nuclease-encoding gene and immunoglobulin loci, and

(2) introducing an antigen-encoding gene into the stock line of animals.

2. Reducing expression of proteins in the animal that are similar to the antigen

In some embodiments, the expression of a protein that is similar (e.g. homologous) or identical to the antigen is reduced or eliminated in the animal at least while the animal is establishing central tolerance. This is advantageous because it reduces or eliminates the establishment of central tolerance against that protein, which, in view of the similarity between the antigen and that protein, may otherwise reduce or eliminate the subsequent immune response to immunisation with the antigen.

Accordingly, in one aspect, the method of the invention comprises:

(a) reducing or eliminating expression of a protein that is similar (e.g. homologous) or identical to the antigen in the animal,

(b) immunising the animal with the antigen, and

wherein step (a) is performed during the period that the animal is establishing central tolerance.

In some embodiments, step (a) is performed during the day, the two days, the three days, the four days, the five days, the six days, the seven days, the eight days, the nine days, the ten days, the eleven days, the twelve days, the thirteen days, the fourteen days, the fifteen days, the sixteen days, the seventeen days, the eighteen days, the nineteen days, the twenty days, the twenty-one days, the twenty-two days, the twenty-three days, the twenty-four days, the twenty-five days, the twenty-six days, the twenty seven days or the twenty-eight days after birth or hatching.

In some embodiments, step (a) is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until no more than four weeks after birth or hatching. In some embodiments, step (a) is performed from fertilisation until no more than three weeks after birth or hatching. In some embodiments, step (a) is performed from fertilisation until no more than two weeks after birth or hatching. In some embodiments, step (a) is performed from fertilisation until no more than one week after birth or hatching.

In some embodiments, step (a) is performed in the period from embryo implantation until central tolerance is established. In some preferred embodiments, step (a) is performed from implantation until no more than four weeks after birth. In some embodiments, step (a) is performed from implantation until no more than three weeks after birth. In some embodiments, step (a) is performed from implantation until no more than two weeks after birth. In some embodiments, step (a) is performed from implantation until no more than one week after birth.

In some embodiments, step (a) is performed during gestation until central tolerance is established. In some preferred embodiments, step (a) is performed during gestation until no more than four weeks after birth. In some embodiments, step (a) is performed during gestation until no more than three weeks after birth. In some embodiments, step (a) is performed during gestation until no more than two weeks after birth. In some embodiments, step (a) is performed during gestation until no more than one week after birth.

In some embodiments, step (a) is performed for no more than one day, for no more than two days, for no more than three days, for no more than four days, for no more than five days, for no more than six days, for no more than seven days, for no more than eight days, for no more than nine days, for no more than ten days, for no more than eleven days, for no more than twelve days, for no more than thirteen days, for no more than fourteen days, for no more than fifteen days, for no more than sixteen days, for no more than seventeen days, for no more than eighteen days, for no more than nineteen days, for no more than twenty days, for no more than twenty-one days, for no more than twenty-two days, for no more than twenty-three days, for no more than twenty-four days, for no more than twenty-five days, for no more than twenty-six days, for no more than twenty seven days or for no more than twenty-eight days.

In some embodiments, step (a) is performed for at least one day, for at least two days, for at least three days, for at least four days, for at least five days, for at least six days, for at least seven days, for at least eight days, for at least nine days, for at least ten days, for at least eleven days, for at least twelve days, for at least thirteen days, for at least fourteen days, for at least fifteen days, for at least sixteen days, for at least seventeen days, for at least eighteen days, for at least nineteen days, for at least twenty days, for at least twenty-one days, for at least twenty-two days, for at least twenty-three days, for at least twenty-four days, for at least twenty-five days, for at least twenty-six days, for at least twenty seven days or for at least twenty-eight days.

In some embodiments, step (a) is performed for the lifetime of the animal. In some embodiments, step (a) is not performed for the lifetime of the animal.

In some embodiments, step (b) is performed after the day, the two days, the three days, the four days, the five days, the six days, the seven days, the eight days, the nine days, the ten days, the eleven days, the twelve days, the thirteen days, the fourteen days, the fifteen days, the sixteen days, the seventeen days, the eighteen days, the nineteen days, the twenty days, the twenty-one days, the twenty-two days, the twenty-three days, the twenty-four days, the twenty-five days, the twenty-six days, the twenty seven days or the twenty-eight days after birth or hatching.

In some embodiments, the animal is a mammal (e.g. a mouse or a rat) and step (b) is performed later than eight weeks after birth. In some embodiments, the animal is a mammal (e.g. a mouse or a rat) and step (b) is performed after weaning.

In some embodiments, step (a) is performed during the central tolerance period and step (b) is performed after the central tolerance period.

In some embodiments, step (a) is performed during the day after birth or hatching and step (b) is performed after the day after birth or hatching. In some embodiments, step (a) is performed during the two days after birth or hatching and step (b) is performed after the two days after birth or hatching. In some embodiments, step (a) is performed during the three days after birth or hatching and step (b) is performed after the three days after birth or hatching. In some embodiments, step (a) is performed during the four days after birth or hatching and step (b) is performed after the four days after birth or hatching. In some embodiments, step (a) is performed during the five days after birth or hatching and step (b) is performed after the five days after birth or hatching. In some embodiments, step (a) is performed during the six days after birth or hatching and step (b) is performed after the six days after birth or hatching. In some embodiments, step (a) is performed during the seven days after birth or hatching and step (b) is performed after the seven days after birth or hatching. In some embodiments, step (a) is performed during the eight days after birth or hatching and step (b) is performed after the eight days after birth or hatching. In some embodiments, step (a) is performed during the nine days after birth or hatching and step (b) is performed after the nine days after birth or hatching. In some embodiments, step (a) is performed during the ten days after birth or hatching and step (b) is performed after the ten days after birth or hatching. In some embodiments, step (a) is performed during the eleven days after birth or hatching and step (b) is performed after the eleven days after birth or hatching. In some embodiments, step (a) is performed during the twelve days after birth or hatching and step (b) is performed after the twelve days after birth or hatching. In some embodiments, step (a) is performed during the thirteen days after birth or hatching and step (b) is performed after the thirteen days after birth or hatching. In some embodiments, step (a) is performed during the fourteen days after birth or hatching and step (b) is performed after the fourteen days after birth or hatching. In some embodiments, step (a) is performed during the fifteen days after birth or hatching and step (b) is performed after the fifteen days after birth or hatching. In some embodiments, step (a) is performed during the sixteen days after birth or hatching and step (b) is performed after the sixteen days after birth or hatching. In some embodiments, step (a) is performed during the seventeen days after birth or hatching and step (b) is performed after the seventeen days after birth or hatching. In some embodiments, step (a) is performed during the eighteen days after birth or hatching and step (b) is performed after the eighteen days after birth or hatching. In some embodiments, step (a) is performed during the nineteen days after birth or hatching and step (b) is performed after the nineteen days after birth or hatching. In some embodiments, step (a) is performed during the twenty days after birth or hatching and step (b) is performed after the twenty days after birth or hatching. In some embodiments, step (a) is performed during the twenty-one days after birth or hatching and step (b) is performed after the twenty-one days after birth or hatching. In some embodiments, step (a) is performed during the twenty-two days after birth or hatching and step (b) is performed after the twenty-two days after birth or hatching. In some embodiments, step (a) is performed during the twenty-three days after birth or hatching and step (b) is performed after the twenty-three days after birth or hatching. In some embodiments, step (a) is performed during the twenty-four days after birth or hatching and step (b) is performed after the twenty-four days after birth or hatching. In some embodiments, step (a) is performed during the twenty-five days after birth or hatching and step (b) is performed after the twenty-five days after birth or hatching. In some embodiments, step (a) is performed during the twenty-six days after birth or hatching and step (b) is performed after the twenty-six days after birth or hatching. In some embodiments, step (a) is performed during the twenty-seven days after birth or hatching and step (b) is performed after the twenty-seven days after birth or hatching. In some embodiments, step (a) is performed during the twenty-eight days after birth or hatching and step (b) is performed after the twenty-eight days after birth or hatching.

In some embodiments, step (a) is performed in fertilised eggs, embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.

In some embodiments, step (a) is performed using Cre-Lox recombination, CRISPR/Cas, homologous recombination, zinc finger nucleases or RNA interference (RNAi) .

In some embodiments, the protein in the animal that is similar or identical to the antigen is not essential for development, and step (a) is performed by removing the gene encoding said protein. In some embodiments, the protein in the animal that is similar or identical to the antigen is not essential for development of a functional immune system, and step (a) is performed by removing the gene encoding said protein.

In some embodiments, an inducible transgene encoding the antigen is introduced into fertilised eggs, ES cells or iPS cells (e.g. by using a viral vector) , and step (b) is performed by inducing antigen expression from the transgene after the animal has established central tolerance. In some embodiments, antigen expression is induced by tetracycline-controlled transcriptional activation.

In some embodiments, an inducible transgene encoding the antigen is introduced into ES cells, the ES cells are injected into blastocysts (e.g. blastocysts that have been treated such that they can only make trophectoderm) , and step (b) is performed by inducing antigen expression from the transgene after the animal has established central tolerance. In some embodiments, antigen expression is induced by tetracycline-controlled transcriptional activation.

In some embodiments, step (b) is performed by injecting the antigen. In some embodiments, the antigen is a protein. In some embodiments, the antigen is a sugar.

In some embodiments, step (b) is performed by immunising the animal with DNA that encodes the antigen. In some embodiments, the gene encoding the antigen is administered in a plasmid. In some embodiments, the gene encoding the antigen is administered in a viral vector. In some embodiments, the plasmid is administered by saline injection or gene gun delivery.

In some embodiments, step (b) is performed by cellular immunisation. Accordingly, in some embodiments, cells expressing the antigen on their surface are administered to the animal. In some preferred embodiments, the cells are allogeneic cells.

In some embodiments, the membrane fraction of cells expressing the antigen on their surface is administered to the animal. In some embodiments, step (b) is performed by immunising the animal with a whole cell extract, cytoplasmic extract or nuclear extract. In some embodiments, the extract is purified. In some embodiments, the extract is obtained from one or more allogeneic animals.

2.1 Mice

In some embodiments, the animal is a mouse and step (a) is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until 1-2 weeks after birth. In some preferred embodiments, step (a) is performed from fertilisation until no more than two weeks after birth. In some preferred embodiments, step (a) is performed from fertilisation until no more than four weeks after birth.

In some embodiments, the animal is a mouse and step (a) is performed in the period from fertilisation until weaning. In some preferred embodiments, step (a) is performed from fertilisation until eight weeks after birth.

In some embodiments, step (a) is performed in the period from embryo implantation until central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until 1-2 weeks after birth. In some preferred embodiments, step (a) is performed from fertilisation until no more than two weeks after birth. In some preferred embodiments, step (a) is performed from embryo implantation until no more than four weeks after birth.

In some embodiments, step (a) is performed in the period from embryo implantation until weaning. In some preferred embodiments, step (a) is performed from embryo implantation until eight weeks after birth.

In some embodiments, step (a) is performed during gestation until central tolerance is established. In some preferred embodiments, step (a) is performed during gestation until 1-2 weeks after birth. In some preferred embodiments, step (a) is performed during gestation until no more than two weeks after birth. In some preferred embodiments, step (a) is performed during gestation until no more than four weeks after birth.

In some embodiments, step (a) is performed during gestation until weaning. In some preferred embodiments, step (a) is performed during gestation until eight weeks after birth.

In some embodiments, the animal is a mouse and step (a) is performed during the fourteen days after birth, during the fifteen days after birth, during the sixteen days after birth, during the seventeen days after birth, or during the eighteen days after birth. In some embodiments, step (a) is performed for the lifetime of the mouse. In some embodiments, step (a) is not performed for the lifetime of the mouse.

In some embodiments, the animal is a mouse and step (a) is performed for no more than the fourteen days after birth, for no more than the fifteen days after birth, for no more than the sixteen days after birth, for no more than the seventeen days after birth, or for no more than the eighteen days after birth.

In some embodiments, the animal is a mouse and step (a) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a mouse and step (a) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a mouse and step (a) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth.

In some embodiments, the animal is a mouse, step (a) is not performed for the lifetime of the mouse, and step (a) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a mouse, step (a) is not performed for the lifetime of the mouse, and step (a) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a mouse, step (a) is not performed for the lifetime of the mouse, and step (a) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth. In some embodiments, the animal is a mouse and step (a) is performed for no more than ten days during the twelve days after birth, or no more than eleven days during the twelve days after birth. In some embodiments, the animal is a mouse and step (a) is performed for no more than ten days during the thirteen days after birth, no more than eleven days during the thirteen days after birth, or no more than twelve days during the thirteen days after birth.

In some embodiments, the animal is a mouse and step (a) is performed for no more than ten days during the fourteen days after birth, no more than eleven days during the fourteen days after birth, no more than twelve days during the fourteen days after birth, or no more than thirteen days during the fourteen days after birth.

In some embodiments, the animal is a mouse and step (a) is performed at least until twelve days after birth, at least until thirteen days after birth, or at least until fourteen days after birth. In some embodiments, step (a) is performed for the lifetime of the mouse.

In some embodiments, the animal is a mouse and step (a) is performed until twelve days after birth, until thirteen days after birth or until fourteen days after birth.

In some embodiments, the animal is a mouse and step (b) is performed after the central tolerance period.

In some embodiments, the animal is a mouse and step (b) is performed after 1-2 weeks after birth.

In some embodiments, the animal is a mouse and step (b) is performed after the twelve days, the thirteen days or the fourteen days after birth.

In some embodiments, the animal is a mouse, step (a) is performed in the period from fertilisation until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until 1-2 weeks after birth, and step (b) is performed after 1-2 weeks after birth. In some preferred embodiments, step (a) is performed from fertilisation until no more than two weeks after birth, and step (b) is performed after two weeks after birth. In some preferred embodiments, step (a) is performed from fertilisation until no more than four weeks after birth, and step (b) is performed after four weeks after birth.

In some embodiments, the animal is a mouse, step (a) is performed in the period from fertilisation until weaning, and step (b) is performed after weaning. In some preferred embodiments, step (a) is performed from fertilisation until eight weeks after birth, and step (b) is performed after eight weeks after birth.

In some embodiments, step (a) is performed in the period from embryo implantation until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed from embryo implantation until 1-2 weeks after birth, and step (b) is performed after 1-2 weeks after birth. In some preferred embodiments, step (a) is performed from embryo implantation until no more than two weeks after birth, and step (b) is performed after two weeks after birth. In some preferred embodiments, step (a) is performed from embryo implantation until no more than four weeks after birth, and step (b) is performed after four weeks after birth.

In some embodiments, the animal is a mouse, step (a) is performed in the period from embryo implantation until weaning, and step (b) is performed after weaning. In some preferred embodiments, step (a) is performed from embryo implantation until eight weeks after birth, and step (b) is performed after eight weeks after birth.

In some embodiments, the animal is a mouse, step (a) is performed during gestation until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed during gestation until 1-2 weeks after birth, and step (b) is performed after 1-2 weeks after birth. In some preferred embodiments, step (a) is performed during gestation until no more than two weeks after birth, and step (b) is performed after two weeks after birth. In some preferred embodiments, step (a) is performed during gestation until no more than four weeks after birth, and step (b) is performed after four weeks after birth.

In some embodiments, the animal is a mouse, step (a) is performed during gestation until weaning, and step (b) is performed after weaning. In some preferred embodiments, step (a) is performed during gestation until eight weeks after birth, and step (b) is performed after eight weeks after birth.

In some embodiments, the animal is a mouse, step (a) is performed for no more than the twelve days after birth and step (b) is performed after the twelve days after birth. In some embodiments, the animal is a mouse, step (a) is performed for no more than the thirteen days after birth and step (b) is performed after the thirteen days after birth. In some embodiments, the animal is a mouse, step (a) is performed for no more than the fourteen days after birth and step (b) is performed after the fourteen days after birth.

2.2 Rats

In some embodiments, the animal is a rat and step (a) is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until 1-2 weeks after birth. In some embodiments, step (a) is performed from fertilisation until no more than two weeks after birth. In some embodiments, step (a) is performed from fertilisation until no more than four weeks after birth.

In some embodiments, the animal is a rat and step (a) is performed in the period from fertilisation until weaning. In some preferred embodiments, step (a) is performed from fertilisation until eight weeks after birth.

In some embodiments, the animal is a rat and step (a) is performed during the fourteen days after birth, during the fifteen days after birth, during the sixteen days after birth, during the seventeen days after birth, or during the eighteen days after birth. In some embodiments, step (a) is performed for the lifetime of the rat. In some embodiments, step (a) is not performed for the lifetime of the rat.

In some embodiments, the animal is a rat and step (a) is performed for no more than the fourteen days after birth, for no more than the fifteen days after birth, for no more than the sixteen days after birth, for no more than the seventeen days after birth, or for no more than the eighteen days after birth.

In some embodiments, the animal is a rat and step (a) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a rat and step (a) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a rat and step (a) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth.

In some embodiments, the animal is a rat, step (a) is not performed for the lifetime of the rat, and step (a) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a rat, step (a) is not performed for the lifetime of the rat, and step (a) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a rat, step (a) is not performed for the lifetime of the rat, and step (a) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth.

In some embodiments, the animal is a rat and step (a) is performed for no more than ten days during the twelve days after birth, or no more than eleven days during the twelve days after birth. In some embodiments, the animal is a rat and step (a) is performed for no more than ten days during the thirteen days after birth, no more than eleven days during the thirteen days after birth, or no more than twelve days during the thirteen days after birth.

In some embodiments, the animal is a rat and step (a) is performed for no more than ten days during the fourteen days after birth, no more than eleven days during the fourteen days after birth, no more than twelve days during the fourteen days after birth, or no more than thirteen days during the fourteen days after birth.

In some embodiments, the animal is a rat and step (a) is performed at least until twelve days after birth, at least until thirteen days after birth, or at least until fourteen days after birth. In some embodiments, step (a) is performed for the lifetime of the rat.

In some embodiments, the animal is a rat and step (a) is performed until twelve days after birth, until thirteen days after birth or until fourteen days after birth.

In some embodiments, the animal is a rat and step (b) is performed after the central tolerance period.

In some embodiments, the animal is a rat and step (b) is performed after 1-2 weeks after birth. In some embodiments, the animal is a rat and step (b) is performed after the twelve days, the thirteen days or the fourteen days after birth.

In some embodiments, the animal is a rat, step (a) is performed in the period from fertilisation until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until 1-2 weeks after birth, and step (b) is performed after 1-2 weeks after birth. In some preferred embodiments, step (a) is performed from fertilisation until no more than two weeks after birth, and step (b) is performed after two weeks after birth. In some preferred embodiments, step (a) is performed from fertilization until no more than four weeks after birth, and step (b) is performed after four weeks after birth.

In some embodiments, the animal is a rat, step (a) is performed in the period from fertilisation until weaning, and step (b) is performed after weaning. In some preferred embodiments, step (a) is performed from fertilisation until eight weeks after birth, and step (b) is performed after eight weeks after birth.

In some embodiments, the animal is a rat, step (a) is performed in the period from embryo implantation until weaning, and step (b) is performed after weaning. In some preferred embodiments, step (a) is performed from embryo implantation until eight weeks after birth, and step (b) is performed after eight weeks after birth.

In some embodiments, the animal is a rat, step (a) is performed during gestation until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed during gestation until 1-2 weeks after birth, and step (b) is performed after 1-2 weeks after birth. In some preferred embodiments, step (a) is performed during gestation until no more than two weeks after birth, and step (b) is performed after two weeks after birth. In some preferred embodiments, step (a) is performed during gestation until no more than four weeks after birth, and step (b) is performed after four weeks after birth.

In some embodiments, the animal is a rat, step (a) is performed during gestation until weaning, and step (b) is performed after weaning. In some preferred embodiments, step (a) is performed during gestation until eight weeks after birth, and step (b) is performed after eight weeks after birth.

In some embodiments, the animal is a rat, step (a) is performed for no more than the twelve days after birth and step (b) is performed after the twelve days after birth. In some embodiments, the animal is a rat, step (a) is performed for no more than the thirteen days after birth and step (b) is performed after the thirteen days after birth. In some embodiments, the animal is a rat, step (a) is performed for no more than the fourteen days after birth and step (b) is performed after the fourteen days after birth.

2.3 Chickens

In some embodiments, the animal is a chicken and step (a) is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until one week, until two weeks, until three weeks or until four weeks after hatching. In some embodiments, step (a) is performed from fertilisation until two weeks after hatching.

In some embodiments, the animal is a chicken and step (a) is performed during incubation until central tolerance is established. In some preferred embodiments, step (a) is performed during incubation until one week, until two weeks, until three weeks or until four weeks after hatching. In some embodiments, step (a) is performed during incubation until two weeks after hatching.

In some embodiments, the animal is a chicken and step (a) is performed from hatching until central tolerance is established. In some preferred embodiments, step (a) is performed from hatching until one week, until two weeks, until three weeks or until four weeks after hatching. In some embodiments, step (a) is performed from hatching until two weeks after hatching.

In some embodiments, the animal is a chicken and step (b) is performed after central tolerance is established.

In some embodiments, the animal is a chicken, step (a) is performed in the period from fertilization until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed from fertilisation until one week after hatching and step (b) is performed after one week after hatching. In some preferred embodiments, step (a) is performed from fertilisation until two weeks after hatching and step (b) is performed after two weeks after hatching. In some preferred embodiments, step (a) is performed from fertilisation until three weeks after hatching and step (b) is performed after three weeks after hatching. In some preferred embodiments, step (a) is performed from fertilisation until four weeks after hatching and step (b) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (a) is performed during incubation until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed during incubation until one week after hatching and step (b) is performed after one week after hatching. In some preferred embodiments, step (a) is performed during incubation until two weeks after hatching and step (b) is performed after two weeks after hatching. In some preferred embodiments, step (a) is performed during incubation until three weeks after hatching and step (b) is performed after three weeks after hatching. In some preferred embodiments, step (a) is performed during incubation until four weeks after hatching and step (b) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (a) is performed after hatching until central tolerance is established, and step (b) is performed after central tolerance is established. In some preferred embodiments, step (a) is performed after hatching until one week after hatching and step (b) is performed after one week after hatching. In some preferred embodiments, step (a) is performed after hatching until two weeks after hatching and step (b) is performed after two weeks after hatching. In some preferred embodiments, step (a) is performed after hatching until three weeks after hatching and step (b) is performed after three weeks after hatching. In some preferred embodiments, step (a) is performed after hatching until four weeks after hatching and step (b) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken and step (a) is performed during the seven days, the eight days, the nine days, the ten days, the eleven days, the twelve days, the thirteen days, the fourteen days, the fifteen days, the sixteen days, the seventeen days, the eighteen days, the nineteen days, the twenty days, the twenty-one days, the twenty-two days, the twenty-three days, the twenty-four days, the twenty-five days, the twenty-six days, the twenty seven days or the twenty-eight days after hatching. In some embodiments, the animal is a chicken and step (a) is performed for the lifetime of the chicken. In some embodiments, the animal is a chicken and step (a) is not performed for the lifetime of the chicken.

In some embodiments, the animal is a chicken and step (a) is performed for no more than the seven days, no more than the eight days, no more than the nine days, no more than the ten days, no more than the eleven days, no more than the twelve days, no more than the thirteen days, no more than the fourteen days, no more than the fifteen days, no more than the sixteen days, no more than the seventeen days, no more than the eighteen days, no more than the nineteen days, no more than the twenty days, no more than the twenty-one days, no more than the twenty-two days, no more than the twenty-three days, no more than the twenty-four days, no more than the twenty-five days, no more than the twenty-six days, no more than the twenty seven days or no more than the twenty-eight days after hatching.

In some embodiments, the animal is a chicken and step (a) is performed for at least one day during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least two days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least three days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least four days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least five days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least six days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least seven days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least eight days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least nine days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least ten days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least eleven days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twelve days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least thirteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least fifteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least sixteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least seventeen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least eighteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least nineteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-one days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-two days during the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-three days during the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-four days during the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-five days during the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-six days during the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-seven days during the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for at least twenty-eight days during the four weeks after hatching.

In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least one day during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least two days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least three days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least four days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least five days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least six days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least seven days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least eight days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least nine days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least ten days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least eleven days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twelve days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least thirteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least fifteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least sixteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least seventeen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least eighteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least nineteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-one days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-two days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-three days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-four days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-five days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-six days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-seven days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, and step (a) is performed for at least twenty-eight days during the four weeks after hatching.

In some embodiments, the animal is a chicken and step (a) is performed for no more than one day during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than two days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than three days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than four days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than five days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than six days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than seven days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than eight days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than nine days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than ten days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than eleven days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than twelve days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than thirteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than fifteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than sixteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than seventeen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than eighteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than nineteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than twenty days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (a) is performed for no more than twenty-one days during the three weeks or the four weeks after hatching.

In some preferred embodiments, the animal is a chicken and step (a) is performed at least until one week, at least until two weeks, at least until three weeks or at least until four weeks after hatching. In some embodiments, step (a) is performed for the lifetime of the chicken.

In some preferred embodiments, the animal is a chicken and step (a) is performed until one week, until two weeks, until three weeks or until four weeks after hatching.

In some embodiments, the animal is a chicken and step (b) is performed after one week, two weeks, three weeks or four weeks after hatching.

In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, step (a) is performed for at least one week after hatching and step (b) is performed after one week after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, step (a) is performed for at least three weeks after hatching and step (b) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, step (a) is performed for at least three weeks after hatching and step (b) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (a) is not performed for the lifetime of the chicken, step (a) is performed for at least four weeks after hatching and step (b) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (a) is performed for no more than one week after hatching and step (b) is performed after one week after hatching. In some embodiments, the animal is a chicken, step (a) is performed for no more than three weeks after hatching and step (b) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (a) is performed for no more than three weeks after hatching and step (b) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (a) is performed for no more than four weeks after hatching and step (b) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (a) is performed during the one week after hatching and step (b) is performed after one week after hatching. In some embodiments, the animal is a chicken, step (a) is performed during the three weeks after hatching and step (b) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (a) is performed during the three weeks after hatching and step (b) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (a) is performed during the four weeks after hatching and step (b) is performed after four weeks after hatching.

3. Reducing or eliminating expression of a protein that is similar or identical to the antigen

In another aspect, the method of the invention comprises:

(A) inducing expression of the antigen in the animal, and

wherein step (A’) is performed preferably before or during the period that the animal is establishing central tolerance,

and wherein step (A) is performed after the animal has established central tolerance.

In some embodiments, step (A') is performed in fertilised eggs, embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.

In some embodiments, step (A’) is performed using Cre-Lox recombination, CRISPR/Cas, homologous recombination, zinc finger nucleases or RNA interference (RNAi) .

In some embodiments, the protein in the animal that is similar or identical to the antigen is not essential for development, and step (A’) is performed by removing the gene encoding said protein.

In some embodiments, the protein in the animal that is similar or identical to the antigen is not essential for development of a functional immune system, and step (A’) is performed by removing the gene encoding said protein.

In some embodiments, an inducible transgene encoding the antigen is introduced into fertilised eggs, ES cells or iPS cells (e.g. by using a viral vector or CRISPR) , and step (A) is performed by inducing antigen expression from the transgene after the animal has established central tolerance. In some embodiments, antigen expression is induced by tetracycline-controlled transcriptional activation.

In some embodiments, an inducible transgene encoding the antigen is introduced into ES cells, the ES cells are injected into blastocysts (e.g. blastocysts that have been treated such that they can only make trophectoderm) , and step (A) is performed by inducing antigen expression from the transgene after the animal has established central tolerance. In some embodiments, antigen expression is induced by tetracycline-controlled transcriptional activation.

In some embodiments, step (A) is performed by immunising the animal with DNA that encodes the antigen. In some embodiments, the gene encoding the antigen is administered in a plasmid. In some embodiments, the plasmid is administered by saline injection or gene gun delivery.

In some embodiments, step (A’) is performed is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until no more than four weeks after birth or hatching. In some preferred embodiments, step (A’) is performed from fertilisation until no more than three weeks after birth or hatching. In some preferred embodiments, step (A’) is performed from fertilisation until no more than two weeks after birth or hatching. In some preferred embodiments, step (A’) is performed from fertilisation until no more than one week after birth or hatching.

In some embodiments, step (A’) is performed in the period from embryo implantation until central tolerance is established. In some preferred embodiments, step (A’) is performed from implantation until no more than four weeks after birth. In some preferred embodiments, step (A’) is performed from implantation until no more than three weeks after birth. In some preferred embodiments, step (A’) is performed from implantation until no more than two weeks after birth. In some preferred embodiments, step (A’) is performed from implantation until no more than one week after birth.

In some embodiments, step (A’) is performed during gestation until central tolerance is established. In some preferred embodiments, step (A’) is performed during gestation until no more than four weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than three weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than two weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than one week after birth.

In some embodiments, step (A’) is performed during the day, the two days, the three days, the four days, the five days, the six days, the seven days, the eight days, the nine days, the ten days, the eleven days, the twelve days, the thirteen days, the fourteen days, the fifteen days, the sixteen days, the seventeen days, the eighteen days, the nineteen days, the twenty days, the twenty-one days, the twenty-two days, the twenty-three days, the twenty-four days, the twenty-five days, the twenty-six days, the twenty seven days or the twenty-eight days after birth or hatching.

In some embodiments, step (A’) is performed for no more than one day, for no more than two days, for no more than three days, for no more than four days, for no more than five days, for no more than six days, for no more than seven days, for no more than eight days, for no more than nine days, for no more than ten days, for no more than eleven days, for no more than twelve days, for no more than thirteen days, for no more than fourteen days, for no more than fifteen days, for no more than sixteen days, for no more than seventeen days, for no more than eighteen days, for no more than nineteen days, for no more than twenty days, for no more than twenty-one days, for no more than twenty-two days, for no more than twenty-three days, for no more than twenty-four days, for no more than twenty-five days, for no more than twenty-six days, for no more than twenty seven days or for no more than twenty-eight days.

In some embodiments, step (A’) is performed until one day, until two days, until three days, until four days, until five days, until six days, until seven days, until eight days, until nine days, until ten days, until eleven days, until twelve days, until thirteen days, until fourteen days, until fifteen days, until sixteen days, until seventeen days, until eighteen days, until nineteen days, until twenty days, until twenty-one days, until twenty-two days, until twenty-three days, until twenty-four days, until twenty-five days, until twenty-six days, until twenty seven days or until twenty-eight days after birth or hatching.

In some embodiments, step (A’) is performed for at least one day, for at least two days, for at least three days, for at least four days, for at least five days, for at least six days, for at least seven days, for at least eight days, for at least nine days, for at least ten days, for at least eleven days, for at least twelve days, for at least thirteen days, for at least fourteen days, for at least fifteen days, for at least sixteen days, for at least seventeen days, for at least eighteen days, for at least nineteen days, for at least twenty days, for at least twenty-one days, for at least twenty-two days, for at least twenty-three days, for at least twenty-four days, for at least twenty-five days, for at least twenty-six days, for at least twenty seven days or for at least twenty-eight days.

In some embodiments, step (A’) is performed for the lifetime of the animal. In some embodiments, step (A’) is not performed for the lifetime of the animal.

In some embodiments, step (A) is performed after the day, the two days, the three days, the four days, the five days, the six days, the seven days, the eight days, the nine days, the ten days, the eleven days, the twelve days, the thirteen days, the fourteen days, the fifteen days, the sixteen days, the seventeen days, the eighteen days, the nineteen days, the twenty days, the twenty-one days, the twenty-two days, the twenty-three days, the twenty-four days, the twenty-five days, the twenty-six days, the twenty seven days or the twenty-eight days after birth or hatching.

In some embodiments, step (A’) is performed during the day after birth or hatching and step (A) is performed after the day after birth or hatching. In some embodiments, step (A’) is performed during the two days after birth or hatching and step (A) is performed after the two days after birth or hatching. In some embodiments, step (A’) is performed during the three days after birth or hatching and step (A) is performed after the three days after birth or hatching. In some embodiments, step (A’) is performed during the four days after birth or hatching and step (A) is performed after the four days after birth or hatching. In some embodiments, step (A’) is performed during the five days after birth or hatching and step (A) is performed after the five days after birth or hatching. In some embodiments, step (A’) is performed during the six days after birth or hatching and step (A) is performed after the six days after birth or hatching. In some embodiments, step (A’) is performed during the seven days after birth or hatching and step (A) is performed after the seven days after birth or hatching. In some embodiments, step (A’) is performed during the eight days after birth or hatching and step (A) is performed after the eight days after birth or hatching. In some embodiments, step (A’) is performed during the nine days after birth or hatching and step (A) is performed after the nine days after birth or hatching. In some embodiments, step (A’) is performed during the ten days after birth or hatching and step (A) is performed after the ten days after birth or hatching. In some embodiments, step (A’) is performed during the eleven days after birth or hatching and step (A) is performed after the eleven days after birth or hatching. In some embodiments, step (A’) is performed during the twelve days after birth or hatching and step (A) is performed after the twelve days after birth or hatching. In some embodiments, step (A’) is performed during the thirteen days after birth or hatching and step (A) is performed after the thirteen days after birth or hatching. In some embodiments, step (A’) is performed during the fourteen days after birth or hatching and step (A) is performed after the fourteen days after birth or hatching. In some embodiments, step (A’) is performed during the fifteen days after birth or hatching and step (A) is performed after the fifteen days after birth or hatching. In some embodiments, step (A’) is performed during the sixteen days after birth or hatching and step (A) is performed after the sixteen days after birth or hatching. In some embodiments, step (A') is performed during the seventeen days after birth or hatching and step (A) is performed after the seventeen days after birth or hatching. In some embodiments, step (A’) is performed during the eighteen days after birth or hatching and step (A) is performed after the eighteen days after birth or hatching. In some embodiments, step (A’) is performed during the nineteen days after birth or hatching and step (A) is performed after the nineteen days after birth or hatching. In some embodiments, step (A’) is performed during the twenty days after birth or hatching and step (A) is performed after the twenty days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-one days after birth or hatching and step (A) is performed after the twenty-one days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-two days after birth or hatching and step (A) is performed after the twenty-two days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-three days after birth or hatching and step (A) is performed after the twenty-three days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-four days after birth or hatching and step (A) is performed after the twenty-four days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-five days after birth or hatching and step (A) is performed after the twenty-five days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-six days after birth or hatching and step (A) is performed after the twenty-six days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-seven days after birth or hatching and step (A) is performed after the twenty-seven days after birth or hatching. In some embodiments, step (A’) is performed during the twenty-eight days after birth or hatching and step (A) is performed after the twenty-eight days after birth or hatching.

In some embodiments, the animal is a mammal (e.g. a mouse or a rat) and step (A) is performed later than eight weeks after birth. In some embodiments, the animal is a mammal (e.g. a mouse or a rat) and step (A) is performed after weaning.

3.1 Mice

In some embodiments, the animal is a mouse and step (A’) is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than two weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than four weeks after birth.

In some embodiments, the animal is a mouse and step (A’) is performed in the period from fertilisation until weaning. In some preferred embodiments, step (A’) is performed from fertilisation until eight weeks after birth.

In some embodiments, step (A’) is performed in the period from embryo implantation until central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than two weeks after birth. In some preferred embodiments, step (A’) is performed from embryo implantation until no more than four weeks after birth.

In some embodiments, step (A’) is performed in the period from embryo implantation until weaning. In some preferred embodiments, step (A’) is performed from embryo implantation until eight weeks after birth.

In some embodiments, step (A’) is performed during gestation until central tolerance is established. In some preferred embodiments, step (A’) is performed during gestation until 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than two weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than four weeks after birth.

In some embodiments, step (A’) is performed during gestation until weaning. In some preferred embodiments, step (A’) is performed during gestation until eight weeks after birth.

In some embodiments, the animal is a mouse and step (A’) is performed during the fourteen days after birth, during the fifteen days after birth, during the sixteen days after birth, during the seventeen days after birth, or during the eighteen days after birth. In some embodiments, step (A’) is performed for the lifetime of the mouse. In some embodiments, step (A’) is not performed for the lifetime of the mouse.

In some embodiments, the animal is a mouse and step (A’) is performed for no more than the fourteen days after birth, for no more than the fifteen days after birth, for no more than the sixteen days after birth, for no more than the seventeen days after birth, or for no more than the eighteen days after birth.

In some embodiments, the animal is a mouse and step (A’) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a mouse and step (A’) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a mouse and step (A’) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth.

In some embodiments, the animal is a mouse, step (A’) is not performed for the lifetime of the mouse, and step (A’) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a mouse, step (A’) is not performed for the lifetime of the mouse, and step (A’) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a mouse, step (A’) is not performed for the lifetime of the mouse, and step (A’) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth. In some embodiments, the animal is a mouse and step (A’) is performed for no more than ten days during the twelve days after birth, or no more than eleven days during the twelve days after birth.

In some embodiments, the animal is a mouse and step (A’) is performed for no more than ten days during the thirteen days after birth, no more than eleven days during the thirteen days after birth, or no more than twelve days during the thirteen days after birth.

In some embodiments, the animal is a mouse and step (A’) is performed for no more than ten days during the fourteen days after birth, no more than eleven days during the fourteen days after birth, no more than twelve days during the fourteen days after birth, or no more than thirteen days during the fourteen days after birth.

In some embodiments, the animal is a mouse and step (A’) is performed at least until twelve days after birth, at least until thirteen days after birth, or at least until fourteen days after birth. In some embodiments, step (A’) is performed for the lifetime of the mouse.

In some embodiments, the animal is a mouse and step (A’) is performed until twelve days after birth, until thirteen days after birth or until fourteen days after birth.

In some embodiments, the animal is a mouse and step (A) is performed after the central tolerance period.

In some embodiments, the animal is a mouse and step (A) is performed after 1-2 weeks after birth. In some embodiments, the animal is a mouse and step (A) is performed after the twelve days, the thirteen days or the fourteen days after birth.

In some embodiments, the animal is a mouse, step (A’) is performed in the period from fertilisation until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until 1-2 weeks after birth, and step (A) is performed after 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than two weeks after birth, and step (A) is performed after two weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than four weeks after birth, and step (A) is performed after four weeks after birth.

In some embodiments, the animal is a mouse, step (A’) is performed in the period from fertilisation until weaning, and step (A) is performed after weaning. In some preferred embodiments, step (A’) is performed from fertilisation until eight weeks after birth, and step (A) is performed after eight weeks after birth.

In some embodiments, step (A’) is performed in the period from embryo implantation until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed from embryo implantation until 1-2 weeks after birth, and step (A) is performed after 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed from embryo implantation until no more than two weeks after birth, and step (A) is performed after two weeks after birth. In some preferred embodiments, step (A’) is performed from embryo implantation until no more than four weeks after birth, and step (A) is performed after four weeks after birth.

In some embodiments, the animal is a mouse, step (A’) is performed in the period from embryo implantation until weaning, and step (A) is performed after weaning. In some preferred embodiments, step (A’) is performed from embryo implantation until eight weeks after birth, and step (A) is performed after eight weeks after birth.

In some embodiments, the animal is a mouse, step (A’) is performed during gestation until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed during gestation until 1-2 weeks after birth, and step (A) is performed after 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than two weeks after birth, and step (A) is performed after two weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than four weeks after birth, and step (A) is performed after four weeks after birth.

In some embodiments, the animal is a mouse, step (A’) is performed during gestation until weaning, and step (A) is performed after weaning. In some preferred embodiments, step (A’) is performed during gestation until eight weeks after birth, and step (A) is performed after eight weeks after birth.

In some embodiments, the animal is a mouse, step (A’) is performed for no more than the twelve days after birth and step (A) is performed after the twelve days after birth. In some embodiments, the animal is a mouse, step (A’) is performed for no more than the thirteen days after birth and step (A) is performed after the thirteen days after birth. In some embodiments, the animal is a mouse, step (A’) is performed for no more than the fourteen days after birth and step (A) is performed after the fourteen days after birth.

3.2 Rats

In some embodiments, the animal is a rat and step (A’) is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than two weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than four weeks after birth.

In some embodiments, the animal is a rat and step (A’) is performed in the period from fertilisation until weaning. In some preferred embodiments, step (A’) is performed from fertilisation until eight weeks after birth.

In some embodiments, step (A’) is performed in the period from embryo implantation until central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than two weeks after birth. In some preferred embodiments, step (A’) is performed from embryo implantation until no more than four weeks after birth. In some embodiments, step (A’) is performed in the period from embryo implantation until weaning. In some preferred embodiments, step (A’) is performed from embryo implantation until eight weeks after birth.

In some embodiments, the animal is a rat and step (A’) is performed during the fourteen days after birth, during the fifteen days after birth, during the sixteen days after birth, during the seventeen days after birth, or during the eighteen days after birth. In some embodiments, step (A’) is performed for the lifetime of the rat. In some embodiments, step (A’) is not performed for the lifetime of the rat.

In some embodiments, the animal is a rat and step (A’) is performed for no more than the fourteen days after birth, for no more than the fifteen days after birth, for no more than the sixteen days after birth, for no more than the seventeen days after birth, or for no more than the eighteen days after birth.

In some embodiments, the animal is a rat and step (A’) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a rat and step (A’) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a rat and step (A’) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth.

In some embodiments, the animal is a rat, step (A’) is not performed for the lifetime of the rat, and step (A’) is performed for at least ten days during the twelve days after birth, at least eleven days during the twelve days after birth, or for the twelve days after birth.

In some embodiments, the animal is a rat, step (A’) is not performed for the lifetime of the rat, and step (A’) is performed for at least ten days during the thirteen days after birth, at least eleven days during the thirteen days after birth, at least twelve days during the thirteen days after birth, or for the thirteen days after birth.

In some embodiments, the animal is a rat, step (A’) is not performed for the lifetime of the rat, and step (A’) is performed for at least ten days during the fourteen days after birth, at least eleven days during the fourteen days after birth, at least twelve days during the fourteen days after birth, at least thirteen days during the fourteen days after birth, or for the fourteen days after birth.

In some embodiments, the animal is a rat and step (A’) is performed for no more than ten days during the twelve days after birth, or no more than eleven days during the twelve days after birth. In some embodiments, the animal is a rat and step (A’) is performed for no more than ten days during the thirteen days after birth, no more than eleven days during the thirteen days after birth, or no more than twelve days during the thirteen days after birth.

In some embodiments, the animal is a rat and step (A’) is performed for no more than ten days during the fourteen days after birth, no more than eleven days during the fourteen days after birth, no more than twelve days during the fourteen days after birth, or no more than thirteen days during the fourteen days after birth.

In some embodiments, the animal is a rat and step (A’) is performed at least until twelve days after birth, at least until thirteen days after birth, or at least until fourteen days after birth. In some embodiments, step (A’) is performed for the lifetime of the rat.

In some embodiments, the animal is a rat and step (A’) is performed until twelve days after birth, until thirteen days after birth or until fourteen days after birth.

In some embodiments, the animal is a rat and step (A) is performed after the central tolerance period.

In some embodiments, the animal is a rat and step (A) is performed after 1-2 weeks after birth. In some embodiments, the animal is a rat and step (A) is performed after the twelve days, the thirteen days or the fourteen days after birth.

In some embodiments, the animal is a rat, step (A’) is performed in the period from fertilisation until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until 1-2 weeks after birth, and step (A) is performed after 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than two weeks after birth, and step (A) is performed after two weeks after birth. In some preferred embodiments, step (A’) is performed from fertilisation until no more than four weeks after birth, and step (A) is performed after four weeks after birth.

In some embodiments, the animal is a rat, step (A’) is performed in the period from fertilisation until weaning, and step (A) is performed after weaning. In some preferred embodiments, step (A’) is performed from fertilisation until eight weeks after birth, and step (A) is performed after eight weeks after birth.

In some embodiments, the animal is a rat, step (A’) is performed in the period from embryo implantation until weaning, and step (A) is performed after weaning. In some preferred embodiments, step (A’) is performed from embryo implantation until eight weeks after birth, and step (A) is performed after eight weeks after birth.

In some embodiments, the animal is a rat, step (A’) is performed during gestation until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed during gestation until 1-2 weeks after birth, and step (A) is performed after 1-2 weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than two weeks after birth, and step (A) is performed after two weeks after birth. In some preferred embodiments, step (A’) is performed during gestation until no more than four weeks after birth, and step (A) is performed after four weeks after birth.

In some embodiments, the animal is a rat, step (A’) is performed during gestation until weaning, and step (A) is performed after weaning. In some preferred embodiments, step (A’) is performed during gestation until eight weeks after birth, and step (A) is performed after eight weeks after birth.

In some embodiments, the animal is a rat, step (A’) is performed for no more than the twelve days after birth and step (A) is performed after the twelve days after birth. In some embodiments, the animal is a rat, step (A’) is performed for no more than the thirteen days after birth and step (A) is performed after the thirteen days after birth. In some embodiments, the animal is a rat, step (A’) is performed for no more than the fourteen days after birth and step (A) is performed after the fourteen days after birth.

3.3 Chickens

In some embodiments, the animal is a chicken and step (A’) is performed in the period from fertilisation until central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until one week, until two weeks, until three weeks or until four weeks after hatching. In some embodiments, step (A’) is performed from fertilisation until two weeks after hatching.

In some embodiments, the animal is a chicken and step (A’) is performed during incubation until central tolerance is established. In some preferred embodiments, step (A’) is performed during incubation until one week, until two weeks, until three weeks or until four weeks after hatching. In some embodiments, step (A’) is performed during incubation until two weeks after hatching.

In some embodiments, the animal is a chicken and step (A’) is performed from hatching until central tolerance is established. In some preferred embodiments, step (A’) is performed from hatching until one week, until two weeks, until three weeks or until four weeks after hatching. In some embodiments, step (A’) is performed from hatching until two weeks after hatching.

In some embodiments, the animal is a chicken and step (A) is performed after central tolerance is established.

In some embodiments, the animal is a chicken, step (A’) is performed in the period from fertilisation until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed from fertilisation until one week after hatching and step (A) is performed after one week after hatching. In some preferred embodiments, step (A’) is performed from fertilisation until two weeks after hatching and step (A) is performed after two weeks after hatching. In some preferred embodiments, step (A’) is performed from fertilisation until three weeks after hatching and step (A) is performed after three weeks after hatching. In some preferred embodiments, step (A’) is performed from fertilisation until four weeks after hatching and step (A) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (A’) is performed during incubation until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed during incubation until one week after hatching and step (A) is performed after one week after hatching. In some preferred embodiments, step (A’) is performed during incubation until two weeks after hatching and step (A) is performed after two weeks after hatching. In some preferred embodiments, step (A’) is performed during incubation until three weeks after hatching and step (A) is performed after three weeks after hatching. In some preferred embodiments, step (A’) is performed during incubation until four weeks after hatching and step (A) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (A’) is performed after hatching until central tolerance is established, and step (A) is performed after central tolerance is established. In some preferred embodiments, step (A’) is performed after hatching until one week after hatching and step (A) is performed after one week after hatching. In some preferred embodiments, step (A’) is performed after hatching until two weeks after hatching and step (A) is performed after two weeks after hatching. In some preferred embodiments, step (A’) is performed after hatching until three weeks after hatching and step (A) is performed after three weeks after hatching. In some preferred embodiments, step (A’) is performed after hatching until four weeks after hatching and step (A) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken and step (A’) is performed during the seven days, the eight days, the nine days, the ten days, the eleven days, the twelve days, the thirteen days, the fourteen days, the fifteen days, the sixteen days, the seventeen days, the eighteen days, the nineteen days, the twenty days, the twenty-one days, the twenty-two days, the twenty-three days, the twenty-four days, the twenty-five days, the twenty-six days, the twenty seven days or the twenty-eight days after birth or hatching. In some embodiments, the animal is a chicken and step (A’) is performed for the lifetime of the chicken. In some embodiments, the animal is a chicken and step (A’) is not performed for the lifetime of the chicken.

In some embodiments, the animal is a chicken and step (A’) is performed for no more than the seven days, no more than the eight days, no more than the nine days, no more than the ten days, no more than the eleven days, no more than the twelve days, no more than the thirteen days, no more than the fourteen days, no more than the fifteen days, no more than the sixteen days, no more than the seventeen days, no more than the eighteen days, no more than the nineteen days, no more than the twenty days, no more than the twenty-one days, no more than the twenty-two days, no more than the twenty-three days, no more than the twenty-four days, no more than the twenty-five days, no more than the twenty-six days, no more than the twenty seven days or no more than the twenty-eight days after hatching.

In some embodiments, the animal is a chicken and step (A’) is performed for at least one day during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least two days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least three days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least four days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least five days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least six days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least seven days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least eight days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least nine days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least ten days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least eleven days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least twelve days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least thirteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A') is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least fifteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least sixteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least seventeen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least eighteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least nineteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least twenty days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for at least twenty-one days during the three weeks or the four weeks after hatching.

In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least one day during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least two days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least three days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least four days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least five days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least six days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least seven days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least eight days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least nine days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least ten days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least eleven days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twelve days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least thirteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least fifteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least sixteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least seventeen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least eighteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least nineteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-one days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-two days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-three days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-four days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-five days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-six days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-seven days during the four weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, and step (A’) is performed for at least twenty-eight days during the four weeks after hatching.

In some embodiments, the animal is a chicken and step (A’) is performed for no more than one day during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than two days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than three days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than four days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than five days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than six days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than seven days during the week, the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than eight days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than nine days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than ten days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than eleven days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than twelve days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than thirteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than fourteen days during the two weeks, the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than fifteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than sixteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than seventeen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than eighteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than nineteen days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than twenty days during the three weeks or the four weeks after hatching. In some embodiments, the animal is a chicken and step (A’) is performed for no more than twenty-one days during the three weeks or the four weeks after hatching.

In some preferred embodiments, the animal is a chicken and step (A’) is performed at least until one week, at least until two weeks, at least until three weeks or at least until four weeks after hatching. In some embodiments, step (A’) is performed for the lifetime of the chicken.

In some preferred embodiments, the animal is a chicken and step (A’) is performed until one week, until two weeks, until three weeks or until four weeks after hatching.

In some embodiments, the animal is a chicken and step (A) is performed after one week, two weeks, three weeks or four weeks after hatching.

In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, step (A’) is performed for at least one week after hatching and step (A) is performed after one week after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, step (A’) is performed for at least three weeks after hatching and step (A) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, step (A’) is performed for at least three weeks after hatching and step (A) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is not performed for the lifetime of the chicken, step (A’) is performed for at least four weeks after hatching and step (A) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (A’) is performed for no more than one week after hatching and step (A) is performed after one week after hatching. In some embodiments, the animal is a chicken, step (A’) is performed for no more than three weeks after hatching and step (A) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is performed for no more than three weeks after hatching and step (A) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is performed for no more than four weeks after hatching and step (A) is performed after four weeks after hatching.

In some embodiments, the animal is a chicken, step (A’) is performed during the one week after hatching and step (A) is performed after one week after hatching. In some embodiments, the animal is a chicken, step (A’) is performed during the three weeks after hatching and step (A) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is performed during the three weeks after hatching and step (A) is performed after two weeks after hatching. In some embodiments, the animal is a chicken, step (A’) is performed during the four weeks after hatching and step (A) is performed after four weeks after hatching.

The following statements relate to both the first and second aspects above.

In some embodiments, the expression of the protein that is similar or identical to the antigen is reduced. In some embodiments, the expression of the protein that is similar or identical to the antigen is eliminated. In some embodiments, the gene encoding the protein that is similar or identical to the antigen is deleted. In some embodiments, the gene encoding for the protein that is similar or identical to the antigen is conditionally deleted. In some embodiments, the gene encoding for the protein that is similar or identical to the antigen is inactivated.

The reduction or elimination of the expression of the protein that is similar or identical to the antigen may be achieved by any appropriate method. Non-limiting examples include gene knockout (e.g. by using homologous recombination, site specific nucleases, zinc-finger nucleases, TALENS or CRISPR) , conditional gene knockout (e.g. by using the Cre-lox recombination system) and gene silencing (e.g. by using antisense oligonucleotides, RNAi, CRISPR or siRNA) .

In some embodiments, the protein that is similar or identical to the antigen comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of the antigen.

In some embodiments, the protein that is similar or identical to the antigen comprises a portion that is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length, wherein the antigen comprises the same portion, e.g. antigenic portion.

In some embodiments, the protein that is similar to the antigen comprises an epitope that is also present in the antigen.

In some embodiments, the protein that is similar to the antigen is capable of being bound by an antibody that specifically binds to the antigen.

In some embodiments, the protein that is similar or identical to the antigen comprises: the entire amino acid sequence of the antigen.

In some embodiments, the protein that is identical to the antigen consists of: the entire amino acid sequence of the antigen.

In some embodiments, the protein that is similar or identical to the antigen is an endogenous protein.

In some embodiments, the method further comprises introducing a gene encoding a replacement protein that has a similar function to the endogenous protein but is not similar in amino acid sequence to the antigen. In some embodiments, reduction or elimination of the endogenous protein prevents normal development of the non-human animal and expression of the replacement protein restores normal development. This is particularly advantageous when the endogenous protein is essential for development of the animal. In some embodiments, the amino acid sequence of the replacement protein is less than 80%identical, less than 70%identical, less than 60%identical or less than 50%identical to the amino acid sequence of the antigen.

In some embodiments, the replacement protein does not comprise an epitope that is present in the antigen.

In some embodiments, the replacement protein comprises a portion that is no more than 3 or no more than 2 amino acids in length, wherein the antigen comprises the same portion. In some embodiments, the replacement protein comprises a portion that is no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length, wherein the antigen comprises the same portion, e.g. antigenic portion.

In some embodiments, the replacement protein is incapable of being bound by an antibody that specifically binds to the antigen.

In some embodiments, the gene encoding the replacement protein is: (i) homologous to the gene encoding the endogenous protein and (ii) sufficiently non-homologous to the antigen, so as to enable the generation of an antigen-specific antibody response following immunisation with the antigen.

In some embodiments, the gene encoding the replacement protein is from the same species as the non-human animal. In some embodiments, the gene encoding the replacement protein is from a different species from that of the non-human animal. For example, in some embodiments, the non-human animal is Mus musculus and the gene encoding the replacement protein is from Mus spretus.

In some embodiments, the gene encoding the replacement protein replaces the gene encoding the endogenous protein in situ.

In some embodiments, the gene encoding the replacement protein is introduced into fertilised eggs, embryonic stem (ES) or induced pluripotent stem (iPS) cells. Any appropriate method can be used to introduce the gene encoding the replacement protein. Non-limiting examples include homologous recombination and the use of site-specific nucleases.

In some embodiments, the gene encoding the replacement protein is introduced into fertilised eggs, ES cells or iPS cells that already contain a transactivator gene.

4. Antigens

In some embodiments, the antigen is a tumour-associated antigen. In some embodiments, the antigen is a tumour-specific antigen. In some embodiments, the antigen is HER2, EGFR, GD2, VEGF (eg. VEGF-A) , VEGFR (eg. VEGFR-2) , PDGFR (eg. PDGFR-α) , CTLA-4, PD-1, PD-L1, RANKL, CD19, CD20, CD33, CD38, CD52, CRH or SLAMF7.

In some embodiments, the antigen is an antigen associated with a pathogen. In some embodiments, the antigen is a viral antigen. In some preferred embodiments, the pathogen is human immunodeficiency virus (HIV) , e.g. HIV-1, an influenza virus strain, human papillomavirus (HPV) , hepatitis C, hepatitis B, hepatitis A or Dengue virus. In some embodiments, the antigen is a bacterial antigen. In some embodiments, the antigen is a protozoan antigen (e.g. a malarial antigen) . In some embodiments, the antigen is a fungal antigen.

In some embodiments, the antigen is BCMA, PSMA or GPC3.

In some embodiments, the antigen is mesothelin (MSLN) . In a preferred embodiment, the antigen is the membrane-bound form of MSLN.

5. Production of antibodies against a particular form of antigen

In some instances, it may be desirable to obtain antibodies that specifically bind to a particular form of the antigen, for example the membrane bound form of a protein, while the same protein is also secreted into circulation. In one example, the non-human animal may be engineered to express the secreted form of the antigen during development but with an inducible switch that changes the secreted form into a membrane bound form. As a result, the non-human animal will be tolerant for the secreted form of the antigen, while induction of the membrane bound form of the antigen after the tolerance period would only elicit an immune response against the membrane bound form of the antigen, but not an immune response against the secreted form. This is illustrated in Figure 5.

In some preferred embodiments, the protein that is similar or identical to the antigen, the expression of which is reduced or eliminated during the period in which the animal is establishing central tolerance, is in a first form, and the expression of the protein in a second form is not reduced or eliminated. In some embodiments, the first and second forms are splice variants. In some embodiments, the first form is a membrane-bound form, the second form is a secreted form, and the antigen is a protein in membrane-bound form. In some embodiments, the first form is a secreted form, the second form is a membrane-bound form and the antigen is a protein in secreted form.

6. Protein complex antigens

In some embodiments, the antigen is a protein complex. In some embodiments, the antigen is a protein complex and the antigen is encoded by more than one gene.

In some embodiments, the antigen is the CD3 co-receptor complex. This complex is present on T cells, contains several CD3 proteins and is encoded by several genes. Induced expression of the CD3 proteins concomitantly in a non-human animal of the invention after the animal has established central tolerance elicits antibodies that recognize the three-dimensional combination of proteins.

7. Non-human animals

The invention provides a transgenic non-human animal that comprises: (a) one or more immunoglobulin loci, (b) a transgene encoding an antigen and (c) (i) a transgene encoding a transactivator that enhances expression of the antigen and/or (c) (ii) an inducible nuclease that enhances expression of the antigen.

The animal used in the methods of the invention is a non-human animal. In some embodiments, the animal is a rodent. In some embodiments, the animal is a rat or a mouse. In some embodiments, the animal is a mouse. In some embodiments, the animal is a rat. In some embodiments, the animal is a chicken. In some embodiments, the animal is a camelid, such as a llama.

In some embodiments, the animal used in the methods of the invention expresses heavy chain-only antibodies. In some embodiments, the animal used in the methods of the invention expresses tetrameric antibodies comprising two heavy chains and two light chains. In some embodiments, the animal used in the methods of the invention expresses both heavy chain-only antibodies and tetrameric antibodies comprising two heavy chains and two light chains.

Transgenic animals may be used in the methods of the invention. Thus, in some embodiments, the animal is a transgenic rodent. In some embodiments, the animal is a transgenic rat or a transgenic mouse. In some embodiments, the animal is a transgenic mouse. In some embodiments, the animal is a transgenic rat. In some embodiments, the animal is a transgenic chicken.

In some embodiments, the transgenic animal used in the methods of the invention expresses heavy chain-only antibodies. In some embodiments, the transgenic animal used in the methods of the invention expresses tetrameric antibodies comprising two heavy and two light chains. In some embodiments, the transgenic animal used in the methods of the invention expresses heavy chain-only antibodies and tetrameric antibodies comprising two heavy and two light chains.

In some embodiments, the transgenic animal used in the methods of the invention comprises one or more transgenic immunoglobulin loci. In some embodiments, one or more endogenous immunoglobulin heavy chain loci in the animal are deleted or silenced and/or one or more endogenous immunoglobulin light chain loci in the animal are deleted or silenced.

In some embodiments, the one or more transgenic immunoglobulin loci comprise: (a) one or more heterologous heavy chain loci, each of which comprises at least one V gene segment, at least one D gene segment, at least one J gene segment and at least one constant region.

In some embodiments, the one or more transgenic immunoglobulin loci comprise: (a) one or more heterologous heavy chain loci, each of which comprises at least one V gene segment, at least one D gene segment, at least one J gene segment and at least one constant region; and (b) one or more heterologous light chain loci, each of which comprises at least one V gene segment, at least one J gene segment and at least one constant region.

In some embodiments, the transgenic non-human animal comprises a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments and human J gene segments operably linked to one or more endogenous constant region gene segments. In some embodiments, the transgenic non-human animal comprises a heterologous immunoglobulin kappa light chain locus comprising human Vκ gene segments and human J gene segments operably linked to one or more endogenous constant region gene segments.

In some embodiments, the transgenic non-human animal comprises a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments and human J gene segments operably linked to one or more endogenous constant region gene segments.

In some embodiments, the transgenic non-human animal comprises:

(i) a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments and human J gene segments operably linked to one or more endogenous constant region gene segments, and

(ii) (a) a heterologous immunoglobulin kappa light chain locus comprising human Vκ gene segments and human J gene segments operably linked to one or more endogenous constant region gene segments, and/or

(ii) (b) a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments and human J gene segments operably linked to one or more endogenous constant region gene segments.

In some embodiments, the transgenic non-human animal is a mammal that comprises:

(i) a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments and human J gene segments operably linked to one or more mammalian constant region gene segments, and

(ii) (a) a heterologous immunoglobulin kappa light chain locus comprising human Vκ gene segments and human J gene segments operably linked to one or more mammalian constant region gene segments, and/or

(ii) (b) a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments and human J gene segments operably linked to one or more mammalian constant region gene segments.

In some embodiments, the transgenic non-human animal is a rodent that comprises:

(i) a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments and human J gene segments operably linked to one or more rodent constant region gene segments, and

(ii) (a) a heterologous immunoglobulin kappa light chain locus comprising human Vκ gene segments and human J gene segments operably linked to one or more rodent constant region gene segments, and/or

(ii) (b) a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments and human J gene segments operably linked to one or more rodent constant region gene segments.

In some embodiments, the transgenic non-human animal is a mouse or rat that comprises:

(i) a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments and human J gene segments operably linked to one or more mouse or rat constant region gene segments, and (ii) (a) a heterologous immunoglobulin kappa light chain locus comprising human Vκ gene segments and human J gene segments operably linked to one or more mouse or rat constant region gene segments, and/or

(ii) (b) a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments and human J gene segments operably linked to one or more mouse or rat constant region gene segments.

In some embodiments, the transgenic non-human animal is a chicken that comprises:

(i) a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments and human J gene segments operably linked to one or more chicken constant region gene segments, and

(ii) (a) a heterologous immunoglobulin kappa light chain locus comprising human VK gene segments and human J gene segments operably linked to one or more chicken constant region gene segments, and/or

(ii) (b) a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments and human J gene segments operably linked to one or more chicken constant region gene segments.

In some embodiments, the heterologous heavy chain locus comprises:

(i) at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten human VH gene segments, and/or

(ii) at least two, at least three, at least four, at least five, or at least six human J segments, and/or

(iii) at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen or at least sixteen human D segments.

In some embodiments, the heterologous heavy chain locus comprises at least four human VH gene segments, six human D gene segments and 19 human D gene segments.

In some embodiments, the transgenic non-human mammal (e.g. mouse) comprises a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments, human J gene segments, and rat constant region gene segments, wherein the human VH gene segments comprise VH1-69, VH4-59, VH3-53, VH3-49, VH4-34, VH3-48, VH3-30, VH3-33, VH3-23, VH1-18, VH3-15, VH4-b, VH1-8, VH3-07, VH2-5, VH4-4, VH1-2, and VH6-1, the human J gene segments comprise six human J gene segments, the human D gene segments comprise 19 human D gene segments and the rat constant region gene segments comprise Cμ, Cy2c, Cy1, Cy2b, and Cα. In some embodiments, the transgenic non-human mammal (e.g. mouse) comprises a heterologous immunoglobulin kappa light chain locus comprising human Vκ gene segments, human Jκ gene segments, and a rat constant region gene segment, wherein the human Vκ gene segments comprise VK2-30, VK2-28, VK1-5, VK1-9, VK1-27, VK1-33, VK1-39, VK3-20, VK3-15, VK3-11, and VK4-1, the human JK gene segments comprise five human JK gene segments, and the constant region gene segment comprises CK.

In some embodiments, the transgenic non-human mammal (e.g. mouse) comprises a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments, human J gene segments, and rat constant region gene segments, wherein the human Vλ gene segments comprise Vλ3-1, Vλ3-19, Vλ2-8, and Vλ1-51, the human J gene segments comprise Jλ1 and Jλ3, and the constant region gene segments comprise Cλ2 and Cλ3.

In some embodiments, the transgenic non-human mammal comprises:

(a) a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments, human J gene segments, and rat constant region gene segments, wherein the human VH gene segments comprise VH1-69, VH4-59, VH3-53, VH3-49, VH4-34, VH3-48, VH3-30, VH3-33, VH3-23, VH1-18, VH3-15, VH4-b, VH1-8, VH3-07, VH2-5, VH4-4, VH1-2, and VH6-1, the human J gene segments comprise six human J gene segments, the human D gene segments comprise 19 human D gene segments and the rat constant region gene segments comprise Cμ, Cγ2c, Cγ1, Cγ2b, and Cα, and

(b) (i) a heterologous immunoglobulin kappa light chain locus comprising human VK gene segments, human JK gene segments, and a rat constant region gene segment, wherein the human VK gene segments comprise VK2-30, VK2-28, VK1-5, VK1-9, VK1-27, VK1-33, VK1-39, VK3-20, VK3-15, VK3-11, and VK4-1, the human JK gene segments comprise five human JK gene segments, and the constant region gene segment comprises CK, and/or (b) (ii) a heterologous immunoglobulin lambda light chain locus comprising human Vλ gene segments, human J gene segments, and rat constant region gene segments, wherein the human Vλ gene segments comprise Vλ3-1, Vλ3-19, Vλ2-8, and Vλ1-51, the human J gene segments comprise Jλ1 and Jλ3, and the constant region gene segments comprise Cλ2 and Cλ3.

In some embodiments, the transgenic non-human animal comprises a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments and human J gene segments operably linked to one or more endogenous constant region gene segments lacking CH1 exon.

In some embodiments, the transgenic non-human animal (e.g. mouse) comprises a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments, human J gene segments, and a mouse constant region gene segment, wherein the human VH gene segments comprise VH3-48, VH3-30, VH3-33, VH3-23, VH3-64, VH3-74, VH3-66, VH3-53, VH6-1, the human D gene segments comprise 27 human D gene segments, the human J gene segments comprise six human J gene segments, and the mouse constant region gene segment comprises Cλ1, lacking CH1.

In some embodiments, the transgenic non-human animal (e.g. mouse) comprises a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments, human J gene segments, and a mouse constant region gene segment, wherein the human VH gene segments comprise VH1-69, VH4-59, VH3-53, VH3-49, VH4-34, VH3-48, VH3-30, VH3-33, VH3-23, VH1-18, VH3-15, VH4-b, VH1-8, VH3-07, VH2-5, VH4-4, VH1-2, and VH6-1, the human J gene segments comprise six human J gene segments, the human D gene segments comprise 19 human D gene segments and the mouse constant region gene segment comprises Cλ1, lacking CH1.

In some embodiments, the transgenic non-human animal (e.g. mouse) comprises a heterologous immunoglobulin heavy chain locus comprising human VH gene segments, human D gene segments, human J gene segments, and mouse constant region gene segments, wherein the human VH gene segments comprise VH3-11, VH3-23, VH3-53, and VH1-46, the human J gene segments comprise six human J gene segments, the human D gene segments comprise 19 human D gene segments and the mouse constant region gene segments comprises Cy2, lacking CH1, and Cy3, lacking CH1.

In some preferred embodiments, the transgenic non-human animal is a humanized mouse, e.g. as described in US 20012/0322108A1, US 2007/0061900A1, US 2011/0258710A1, US 2001/0283376A1, U.S. Pat. No. 6,596,541 or U.S. Pat. No. 7,105,24.

In some embodiments, the transgenic non-human animal is a mouse as described in WO 2016/062990.

In some embodiments, the transgenic non-human animal is a rat as described in WO 2008/151081, WO 2017/223111, WO 2018/039180, WO 2018/052503 or WO 2018/119215.

In some embodiments, the transgenic non-human animal is a mouse as described in WO 2011/0004192, WO 2011/158009, WO 2012/063048, WO 2013/041844, WO 2013/041845, WO2013/041846, WO 2013/045916, WO2013/061098, WO2013/079953, WO2013/144567, WO 2013/144566, WO 2013/171505, WO 2015/040402, WO 2015/049517, WO 2018/011573 or WO 2019/008123.

In some embodiment, the transgenic non-human animal is a chicken (e.g. as described in Ching et al. (2018) MAbs 10 (1) : 71-80) .

The various steps of the methods may be carried out at the same time or at different times, in the same geographical location or in different geographical locations, e.g. countries, and by the same or different people of entities.

Various aspects and embodiments of the invention are described below in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Hybridoma methods

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975) . In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103) Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) , the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ( “HAT medium” ) , which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133: 3001 (1984) ; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63) ) . The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) . Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980) . Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103) . Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Recombinant DNA methods

Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) . The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994) ) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “humanized antibodies” , “human antibodies” , or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) ; and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96) . Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96) .

Production in transgenic plants and fungi

In addition, humanized antibodies can be produced in transgenic plants, as an inexpensive production alternative to existing mammalian systems. For example, the transgenic plant may be a tobacco plant, i.e., Nicotiania benthamiana, and Nicotiana tabaccum. The antibodies are purified from the plant leaves. Stable transformation of the plants can be achieved through the use of Agrobacterium tumefaciens or particle bombardment. For example, nucleic acid expression vectors containing at least the heavy and light chain sequences are expressed in bacterial cultures, i.e., A. tumefaciens strain BLA4404, via transformation. Infiltration of the plants can be accomplished via injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of the antibodies can be readily be performed by many of the methods known to the skilled artisan in the art. Other methods for antibody production in plants are described in, for example, Fischer et al., Vaccine, 2003, 21: 820-5; and Ko et al, Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. As such, the present invention further provides any cell or plant comprising a vector that encodes the antibody of the present invention, or produces the antibody of the present invention.

In addition, an (human) antibody of interest may be produced in fungi. For example, the fungus may be Myceliophthora thermophila (e.g. Myceliophthora thermophila strain C1; Visser et al. (2011) Industrial Biotechnology 7 (3) : 214-223) . Other examples include Aspergillus species (e.g. A. oryzae (Huynh et al. (2020) Fungal Biology and Biotechnology 7: 7) , A. niger (Ward et al. (2004) Environ. Microbiol. 70: 2567-76) , or A. awamori (Joosten et al. (2003) Microb. Cell Fact 2: 1) ) and Trichoderma species (e.g. T. reesei (et al. (1993) Biotechnology 11: 591-595) ) . In other instances, the fungus may be a yeast, such as Saccharomyces cerevisiae, Candida boidinii, Hansenula polymorpha, Pichia methanolica, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces lactis or Ogataea minuta (Joosten et al. (2003) ; Suzuki et al. (2017) J Biosci Bioeng. 124: 156-63) .

Additional available techniques

In addition, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) ) . Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in WO 2006/008548, WO 2007/096779, WO 2010/109165, WO 2010/070263, WO 2014/141189 and WO 2014/141192.

One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. No. 5,916,771. This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.

The antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor) , and a nucleic acid binding moiety (e.g. polylysine) , viral vector (e.g. a DNA or RNA viral vector) , fusion proteins such as described in PCT/US 95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine) , plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64: 487 (1995) ; Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995) ; Geller, A. I. et al., Proc Natl. Acad. Sci. : U.S.A. 90: 7603 (1993) ; Geller, A. I., et al., Proc Natl. Acad. Sci USA 87: 1149 (1990) , Adenovirus Vectors (see LeGal LaSalle et al., Science, 259: 988 (1993) ; Davidson, et al., Nat. Genet 3: 219 (1993) ; Yang, et al., J. Virol. 69: 2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet. 8: 148 (1994) .

Pox viral vectors introduce the gene into the cell cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months) , which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

The vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994) ; Morrison et al., Am. J. Physiol. 266: 292-305 (1994) ) . Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.

These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of MSLN in a sample. The antibody can also be used to try to bind to MSLN and disrupt the interaction between MSLN and MUC16.

In a preferred embodiment, the antibodies of the present invention are full-length antibodies, containing an Fc region similar to wild-type Fc regions that bind to Fc receptors.

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in neutralizing or preventing viral infection. For example, cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) . (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992) ) . Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989) ) . In a preferred embodiment, the antibody of the present invention has modifications of the Fc region, such that the Fc region does not bind to the Fc receptors. Preferably, the Fc receptor is Fcγ receptor. Particularly preferred are antibodies with modification of the Fc region such that the Fc region does not bind to Fcγ, but still binds to neonatal Fc receptor.

The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) , or a radioactive isotope (i.e., a radioconjugate) .

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa) , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I 131In, 90Y, and 186Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP) , iminothiolane (IT) , bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL) , active esters (such as disuccinimidyl suberate) , aldehydes (such as glutareldehyde) , bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine) , bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine) , diisocyanates (such as tolyene 2, 6-diisocyanate) , and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) . For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987) . Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94/11026) . Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. (See, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds) , Carger Press, New York, (1989) , the entire contents of which are incorporated herein by reference) .

Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun 133: 1335-2549 (1984) ; Jansen et al, Immunological Reviews 62: 185-216 (1982) ; and Vitetta et al., Science 238: 1098 (1987) ) . Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44: 201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester) . See also, U.S. Pat. No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha- (2-pridyl-dithio) -toluene (Pierce Chem. Co., Cat. (21558G) ; (iii) SPDP (succinimidyl-6 [3- (2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat #21651G) ; (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3- (2-pyridyldithio) -propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G) ; and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985) ; Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980) ; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE) . Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab′fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

Therapeutic Methods

The antibodies provide herein can be administered to slow or inhibit the progression of a mesothelin-positive cancer, or inhibit the metastasis of a mesothelin-positive cancer. In these applications, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a cancer that expresses mesothelin, such as mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer or ovarian cancer.

Provided herein is a method of treating a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an antibody described herein. Also provided herein is a method of inhibiting metastasis of a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an antibody described herein. In some embodiments, the mesothelin-positive cancer is mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer or ovarian cancer.

Administration of an antibody disclosed herein can also be accompanied by administration of other anti-cancer agents or therapeutic treatments (such as surgical resection of a tumor) . Any suitable anti-cancer agent can be administered in combination with the antibodies disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

Another common treatment for some types of cancer is surgical treatment, for example surgical resection of a metastatic tumor. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.

Methods for diagnosis and detection

Methods are provided herein for detecting mesothelin protein in vitro or in vivo. In some cases, mesothelin expression is detected in a biological sample. The sample can be any sample, including, but not limited to, blood samples, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.

Provided herein is a method of determining if a subject has a mesothelin-positive cancer by contacting a sample from the subject with a mesothelin-specific monoclonal antibody disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having a mesothelin-positive cancer.

In another embodiment, provided is a method of confirming a diagnosis of a mesothelin-positive cancer in a subject by contacting a sample from a subject diagnosed with a mesothelin-positive cancer with a mesothelin-specific monoclonal antibody disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms the diagnosis of a mesothelin-positive cancer in the subject.

In some examples of the disclosed methods, the monoclonal antibody is directly labeled.

In other examples, the methods further include contacting a second antibody that specifically binds the monoclonal antibody with the sample; and detecting the binding of the second antibody. An increase in binding of the second antibody to the sample as compared to binding of the second antibody to a control sample detects a mesothelin-positive cancer in the subject or confirms the diagnosis of a mesothelin-positive cancer in the subject.

In some cases, the cancer is mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer or ovarian cancer.

In some examples, the control sample is a sample from a subject without cancer. In particular examples, the sample is a blood or tissue sample.

In some embodiments of the methods of diagnosis and detection, the anti-MSLN antibody is directly labeled with a detectable label. In another embodiment, the anti-MSLN antibody (the first antibody) is unlabeled and a second antibody or other molecule that can bind the first is labeled. As is well known to one of skill in the art, a secondary antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, mesothelin can be assayed in a biological sample by a competition immunoassay utilizing mesothelin protein standards labeled with a detectable substance and an unlabeled anti-MSLN antibody. In this assay, the biological sample, the labeled MSLN protein standards and the anti-MSLN antibody are combined and the amount of labeled MSLN protein standard bound to the unlabeled antibody is determined. The amount of MSLN in the biological sample is inversely proportional to the amount of labeled MSLN protein standard bound to the anti-MSLN antibody.

The immunoassays and methods disclosed herein can be used for a number of purposes. In one embodiment, the anti-MSLN antibody may be used to detect the production of MSLN in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of MSLN in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, the MSLN is cell-surface MSLN. In other examples, the MSLN protein is soluble (e.g. in a cell culture supernatant or in a body fluid sample, such as a blood or serum sample) .

In one embodiment, a kit is provided for detecting MSLN in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Kits for detecting a polypeptide will typically comprise a monoclonal anti-MSLN antibody, such as any of the monoclonal antibodies disclosed herein. In a further embodiment, the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label) .

In one embodiment, a kit includes instructional materials disclosing means of use of an anti-MSLN antibody. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files) . The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like) . The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting MSLN in a biological sample generally includes the steps of contacting the biological sample with an anti-MSLN antibody. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

The antibodies disclosed herein can also be utilized in immunoassays, such as, but not limited to radioimmunoassays (RIAs) , ELISA, or immunohistochemical assays. The antibodies can also be used for fluorescence activated cell sorting (FACS) . FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5,061,620) . Any of the monoclonal antibodies that bind mesothelin, as disclosed herein, can be used in these assays. Thus, the antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.

EXAMPLES

EXAMPLE 1. ANIMAL IMMUNIZATION SCHEMES

In order to obtain MSLN-specific antibodies, Harbour H2L2 or HCAb transgenic mice (https: //harbourantibodies. com/) were immunised through different approaches. These immunisations yielded a number of H2L2 and HCAb antibodies that bind MSLN extracellular (ECD) proteins and MSLN-expressing cells such as COV644 cells (an ovarian epithelial-mucinous carcinoma cell line expressing MSLN) .

1.1 immunization by injection of MSLN proteins

Recombinant human MSLN ECD His-tag protein (Acro Biosystem, Catalog #MSN-H5223) or recombinant cynomolgus MSLN ECD His-tag protein (with the sequence set forth below, SEQ ID NO: 277) was used as the immunogen to immunize Harbour H2L2 or HCAb transgenic mice (https: //harbourantibodies. com/) .

Cynomolgus MSLN ECD His-tag protein (SEQ ID NO: 277)

An example of immunization schemes for Harbour H2L2 mice immunization cohorts is listed in Table 7 below. In brief, each mouse was administrated with 50 μg of the immunogen for the first boost and 25 μg for following boosts via s. c. or i. p. together with adjuvant (Sigma, S6322) . The immunization was conducted bi-weekly for a total of 6 times. Final immunization was conducted with immunogen diluted in PBS via i. p.. Serum titers were tested against human Recombinant human MSLN ECD His-tag protein (Acro Biosystem, Catalog #MSN-H5223) using ELISA and FACS.

In another example, immunization of Harbour HCAb mice adopts similar immunization schemes.

An example of the scheme for generating anti-mesothelin H2L2 antibodies by antigen immunization and hybridoma clone screening is illustrated in Figure 1.

Table 7. immunization schemes

1.2 immunization by induced antigen expression

In this example, the immune response against target antigen was raised by immunization with induced expression of the antigen in mice without exogenous injected antigens. Harbour H2L2 or HCAb transgenic mice (https: //harbourantibodies. com/) were used in this example. Figure 2 illustrates the exemplified scheme of generating anti-mesothelin HCAb antibodies by immunizing Harbour HCAb transgenic mice (https: //harbourantibodies. com/) by induction of human mesothelin expression via transgenesis in the mice post weaning, followed by HEK293-pCAG-HCAb library generation and screening to identify MSLN-specific HCAb.

The examples of engineering rodents to obtain inducible immunization are shown in Figure 3. Figure 4 shows examples of engineering rodents to obtain immunization against antigen which has a very similar or identical endogenous protein in the rodent. Additional variations like in Figure 3 and more are also possible. Figure 5 shows exemplary scheme for engineering the secreted versus membrane-bound form of the protein may be done in a different order or in different ways as in Figure 3 and more.

1.2.1. Proof of principle in H2L2 mice

In this example H2L2 mice were used (WO 2014/141189) . The endogenous heavy and light chain immunoglobulin locus are inactivated in these mice. These were further engineered using microinjection of fertilised eggs to introduce a transgene. The transgene contained a commercially available Tet-responsive element (TRE) -minimal CMV promoter coupled to the coding region of the human HER2 gene coding for the Erb-B2 Receptor Tyrosine Kinase 2 protein (e.g. https: //www. genecards. org/cgi-bin/carddisp. pl? gene=ERBB2) , which is frequently expressed on breast tumours and a target antigen for antibody mediated therapy. In particular, the ERBB2 gene was cloned into a pTRE-Tight vector (ClonTech 631059) , which is shown in Figure 6.

The generation of RNPA2B1-rtTA mice is discussed in Katsantoni (2007) BMC Dev Biol. 7: 108. These mice were crossed with the H2L2 mice to generate a stock line for future purposes (panel A of Figure 7) . The resulting rtTA/H2L2 mice were used to obtain fertilised eggs for microinjection of the TRE-HER2 gene resulting in mice that ubiquitously express rtTA, have a silent TRE-HER2 gene and a background of H2L2 immunoglobulin loci (panel B of Figure 7) .

Doxycycline is administered via drinking water (1 gram/litre) starting from 8 weeks after birth, i.e. long after the establishment of central tolerance in the mice. The addition of doxycycline results in the expression of HER2 protein being induced (panel B of Figure 7) . The induction was carried out 4 times for 3 days in 2-week intervals. After this immunisation period, the serum from the mice was analysed by ELISA and the results were plotted as a dilution series (panel C of Figure 7) . As shown in the figure, 5 (designated as 226, 227, 340, 18-415, and 208) out of 8 H2L2 mice on doxycycline showed a specific response to HER2 as evidence by the presence of antibodies against HER2 antigen in the serum.

1.2.2. Proof of principle in HCAb mice

In this example, two lines of HCAb mice (8V3 line and 9V3 line) disclosed in WO 2014/141192 were used. The 8V3 line contains eight VH3 genes, while the 9V3 line carries nine VH3 genes. Both 8V3 line and 9V3 line have their endogenous heavy chain immunoglobulin locus inactivated. These mice were further engineered using microinjection of fertilised eggs to introduce the two transgenes described in 1.2.1 section.

Specifically, the HCAb and RNPA2B1-rtTA mice were crossed together to generate a stock line for future purposes (panel A of Figure 8) . The resulting rtTA/HCAb mice were used to obtained fertilised eggs for micro injection of the TRE-HER2 gene resulting in mice that ubiquitously express rtTA, have a silent TRE-HER2 gene and a background of H2L2 immunoglobulin loci (panel B of Figure 8) . Doxycycline is administered via drinking water (1 gram/litre) starting 8 weeks after birth, i.e. long after the establishment of central tolerance in the mice. The addition of doxycycline results in the expression of HER2 protein being induced (panel B of Figure 8) . The induction was carried out 4 times for 3 days in 2 week intervals.

After this immunization period the serum was analysed by ELISA and the results were plotted as a dilution series (panel C of Figure 8) . 4 out of 8 of the 8V3 mice and 5 out of 5 of the 9V3 mice on doxycycline showed a specific response to HER2 as evidence by the presence of antibodies against HER2 antigen in the serum.

1.2.3 Inducting human MSLN expression in HCAb mice

This example shows the same procedure as carried out in 1.2.2 section, but for a different antigen in an effort to obtain an antibody recognizing the membrane bound form of the antigen rather than its soluble form that is not membrane bound. HCAb 9V3 mice were generated that carried the human mesothelin (MSLN) gene rather than Her2 as shown in 1.2.2 section. After immunisation, the serum was analysed by ELISA and the results were plotted as a dilution series. Two out of seven mice scored positive (Figure 9) . Mesothelin is expressed as a membrane protein on a number of tumours, but also appears as a soluble protein in circulation after cleavage of the membrane. Further analysis showed that some of the resulting HCAbs bind the membrane rather than soluble form of mesothelin.

1.2.4 Anti-mesothelin HCAbs

In order to obtain anti-membrane bound MSLN antibodies, HCAb transgenic miceTM (https: //harbourantibodies. com/) were immunised by induction of human mesothelin expression via transgenesis in the mice post weaning to generate heavy chain-only antibodies (HCAb) . These immunisations yielded a number of HCAb candidates that bind MSLN on COV644 cells (an ovarian epithelial-mucinous carcinoma cell line expressing MSLN) or human mesothelin transfected and expressed on CHO cells (aChinese hamster ovary cell line) .

The HCAb immunization and generation was carried out after pulldown of the antibody positive cells by standard methods as described in GB059171 and EP2411408. For HCAb both induction of human mesothelin in HCAb mice and antigen injection were used. These immunisations yielded a number of antibodies, which were tested in a standard ELISA assay using mesothelin as the antigen and tested in a cell-based ELISA using COV644 cells. This yielded HCAbs: 19G6, 51 8-10, 141pl1-4, 11pl1-4, 11A10, 2E12, 7D7-1, 7F8, 9E4, 10E3, 14E8, 14A8, 16G1, 17F6, 20B8, PR005536, PR005537, PR005541, PR005542, PR005545, PR005548, PR004198, PR004199. These HCAbs were subdivided into two groups: those that bind the membrane bound form of mesothelin and those that block the interaction between MSLN and MUC16. Those that bind the membrane bound form of mesothelin were obtained by induction of human mesothelin in HCAb mice.

To further characterize this distinction, 30 nM of the candidate HCAbs were tested by FACS and bio-layer inferometry (Octet bioForte) for binding to live COV644 cells expressing mesothelin in the presence or absence of 0 to 60nM soluble mesothelin (see, Example 8 and Figures 21, 22 and 23) .

Antibodies binding the membrane bound form of MSLN can be advantageous for diagnostic and therapeutic purposes because they will have a lower background for imaging because they do not bind soluble MSLN and can be used at a lower dose for therapeutic purposes because they do not bind soluble MSLN.

EXAMPLE 2. SCREENING FOR MSLN-SPECIFIC ANTIBODIES

2.1 hybridoma generation and screening for H2L2 antibodies

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975) . In a hybridoma method, lymphocytes are then fused with an immortalized cell line to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103) . Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. The antibody supernatants produced by the hybridoma cells can be screened for the binding specificity to the target by in vitro assays such as enzyme-linked immunoabsorbent assay (ELISA) . After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103) .

An exemplified scheme of generating anti-mesothelin H2L2 antibodies by antigen immunization and hybridoma clone screening is illustrated in Figure 1.

2.2 single B-cell screening for H2L2 antibodies

The Optofluidic system was used for single B cell screening. The system uses optical-electric positioning (OEPTM) technology to move individual cells, and allow simultaneous biological function tests, experimental analysis, positive clone selection and other operations under cell culture conditions. The Beacon platform can perform these tasks in a massively parallel, automated manner on thousands of cells.

In this example, a plasma cell discovery workflow was used. In each experiment, up to 14,000 individual plasma cells were screened for secretion of MSLN-specific antibodies. Then, plasma cells that secreted antigen-specific antibodies were transferred to 96-well plates for subsequent single B cell sequencing to identify the heavy chain and light chain of the antibody produced by a single B cell (monoclonal) . Figure 10 shows the screening strategy and process.

The example used a single B cell sequencing method to obtain the sequences of heavy chain and light chain of the antibody from a single plasma cell. General procedures include extraction and purification of the total RNA from single plasma cell lysate, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification of the DNA sequences encoding heavy and light chains of an antibody, cloning and transfection, and Sanger sequencing. Uniqueness and cluster analysis on the obtained sequences was performed, and then DNA sequences encoding the paired heavy and light chain of the antibody were synthesized.

2.3 HEK293-pCAG-HCAb directed cloning screening for HCAb antibodies

In this example, lymph nodes from mice with high antibody titers were harvested to prepare cDNA. The variable regions of HCAb cDNA were amplified by PCR using specific primers (5’ -GGTGTCCAGTGTSAGGTGCAGCTG-3’ (SEQ ID NO: 278) , 5’ -AATCCCTGGGCACTGAAGAGACGGTGACC-3’ (SEQ ID NO: 279) ) and cloned on mammalian expression vector (pCAG) which contains human immunoglobulin heavy chain Fc part of the IgG1 subclass, named as pCAG-HCAb libraries. The plasmids of pCAG-HCAb libraries were prepared and transfected into HEK293 cells (ATCC, CRL-1573) on 96-well plates for expression, then the supernatants of HEK293-pCAG-HCAb were harvested and transferred to different 96-well plates for screening by in vitro binding assay. Binding to stable cell line CHOK1-huMSLN (Kyinno, KC-1152) expressing human MSLN, and to stable cell line CHOK1-cynoMSLN (Kyinno, KC-1174) expressing cynomolgus monkey MSLN were tested by Mirrorball (SPT Labtech) . HEK293 cell supernatants which exhibited binding to both CHOK1-huMSLN and CHOK1-cynoMSLN were selected for subsequently FACS screening. Finally, multiple HCAb clones were selected for the further characterization.

Figure 2 illustrates the exemplified scheme of generating anti-mesothelin HCAb antibodies by immunizing Harbour HCAb mice by induction of human mesothelin expression via transgenesis in the mice post weaning, followed by HEK293-pCAG-HCAb library generation and screening to identify MSLN-specific HCAb.

EXAMPLE 3. ANTIBODY PRODUCTION AND PURIFICATION

The recombinant plasmids encoding target antibodies were transiently transfected into HEK293-6E cells or 293-F cells using PEI (Polyscience, 24885) . After transfection, the cells were incubated at 37℃ with 5%CO₂ and shaking at 120 rpm. The cell culture supernatants containing target antibodies were harvested 6-7 days post transfection by centrifugation and filtration. Monoclonal antibodies were purified using Protein A magnetic beads (AmMag Protein A Magnetic Beads, Genscript, L00695) .

The purity of the antibodies was tested by SEC-HPLC (Agilent 1260 Infinity II HPLC with Welch Xtimate SEC-300 Colum, 1 X PBS pH 7.4 as mobile phase) and SDS-PAGE (SurePAGE, Bis-Tris, 10x8, 4-12%, 12 wells, Genscript, M00653) . Recombinant antibodies were successfully expressed and purified for further characterization.

The amino acid sequences of the antibodies were listed in Tables 1 -6 above.

In the meantime, anti-MSLN antibody Amatuximab was also produced following the procedures showed above with sequence information from IMGT (http: //www. imgt. org/3Dstructure-DB/cgi/details. cgi? pdbcode=9343) . This antibody was used as a control in subsequent studies and assigned a code of PR000685.

Example 4. Binding Activity of Antibodies to MSLN Expressing cells

Binding of recombinant anti-MSLN antibodies to human or cynomolgus MSLN-expressing cells was tested by flow cytometry. In this example, MSLN-expressing cell lines are CHOK1 cell lines that had been transfected to express human MSLN on the surface (CHOK1-hu MSLN, vendor: Kyinno, catalog: KC-1152) or cynomolgus MSLN (CHOK1-cyno MSLN, vendor: Kyinno, catalog: KC-1174) , as well as COV644 cell line (ECACC, catalog: 07071908) .

In brief, anti-MSLN antibodies were serially diluted in staining buffer (PBS containing 2%FBS) . 50 μL of diluted antibody solution was added to 50 μL of cell suspension containing 1-2 ×10⁵ cells and incubated at 4℃ for 1 hour. The cells were washed twice with staining buffer (PBS containing 2%FBS) , and 100 μL of 1: 1000 diluted florescent labeled anti-human IgG antibody (Alexa488 AffiniPure Goat Anti-Human IgG (H+L) , Jackson ImmunoResearch, Catalog 109-545-088) was added into each well. After 1-hour incubation at 4℃, cells were washed twice with staining buffer and subjected to flow cytometry. PR000685 (Amatuximab) and non-relevant IgG isotype control (Crownbio) were used as positive and negative controls respectively.

The results for H2L2 antibodies are shown in Figures 11-13 and Table 8 below. The results indicate that H2L2 antibodies including PR300147, PR300162, PR300163, PR300187, PR300193, PR300281, PR300283, PR300284 and PR300286 showed strong binding activity to both human and cynomolgus MSLN expressing cells, with EC50 values comparable to PR000685 (Amatuximab) . These results indicate that the anti-MSLN H2L2 antibodies are capable of binding to human and cynomolgus MSLN on cell membrane with high affinity.

Table 8. Binding of anti-MSLN H2L2 antibodies to cell surface MSLN by FACS

The results for HCAb antibodies are shown in Figures 14-15 and Tables 9-10. As shown in Figure 14and Table 9, HCAb antibodies including PR005536, PR005537, PR005541, PR005542, PR005545, PR005548, PR004197 also showed strong binding to both human and cynomolgus MSLN overexpressing cells, with EC50 values and/or binding MFI top values comparable to PR000685 (Amatuximab) . As shown in Figure 15 and Table 10, HCAb antibodies PR004198 and PR004199 showed binding to human MSLN overexpressing cells but no binding to cynomolgus MSLN overexpressing cells.

Table 9. Binding of anti-MSLN HCAb antibodies to cell surface MSLN by FACS

Table 10. Binding of anti-MSLN HCAb antibodies to cell surface MSLN by FACS

Example 5. Binding Activity of Antibodies to Soluble MSLN Protein by BLI Method

In the example, binding kinetics of anti-MSLN antibodies to soluble MSLN were analyzed by Octet Red96e (Fortebio) . In Bio-Layer Interferometry (BLI) analysis, recombinant human MSLN-His tag (Acro Biosystems, Catalog #MSN-H5223) was serially diluted with 1× kinetic buffer (Fortebio) . Anti-MSLN antibodies were diluted to 5 μg/mL. Then the diluted antibodies, antigen and regeneration buffer (10 mM glycine pH 1.75) were added to 96-well plates (Greiner) . Rate constants for association and dissociation were measured using AHC sensor (Fortebio) . The sensor surface was regenerated after each binding experiment with regeneration buffer. The traces were processed using Octet Data Analysis Software (version 11.0, Pall ForteBio, CA, USA) . The K_D values of the binding of the H2L2 antibodies against soluble human MSLN are summarized in Table 11, and the results for HCAb antibodies are shown in Table 12.

As shown in Table 11, in Octect analysis, the H2L2 antibodies showed moderate or relative low binding affinity to soluble MSLN, with the K_D values of most of the antibodies higher than 1nM and up to around 100nM. Together with the results in Example 4, it is indicated that all the antibodies showed low binding activity to soluble MSLN while preserving higher binding activity to membrane-bound MSLN.

These anti-MSLN H2L2 antibodies show weaker binding affinity to soluble MSLN compare to PR000685 (Amatuximab) , which is advantageous when applied as therapeutic antibodies since the antibodies would preferably bind to cell surface MSLN on tumor cells rather than soluble MSLN in circulation system. Similar results were observed for HCAb antibodies (Table 12) .

Table 11. Binding of anti-MSLN H2L2 antibodies to soluble human MSLN

Table 12. Binding of anti-MSLN HCAb antibodies to soluble human MSLN

Example 6. Epitope Binning of Antibodies

In epitope binning analysis using Octet, antibodies were tested against each other in a pairwise fashion to see whether a first antibody blocks a second antibody’s binding to the epitope of the antigen. The first and second antibodies were diluted to the same concentration which saturates the binding of immobilized antigens. Recombinant MSLN-His was biotinylationed and then immobilized to SA sensor as antigen. The first antibody was incubated with the immobilized antigen, followed by addition of the second antibody. Data were analyzed by Octet Data Analysis Software. The rate of RU value of an antibody when used as the second antibody to that when used as the first antibody was calculated as the co-binding rate of this antibody to other antibodies. Generally speaking, a rate >50% (e.g. near 100%) indicates the two antibodies binds to different epitope bins; a rate <20%indicates an overlapping bin of two assessed antibodies.

The co-binding rates of H2L2 antibodies are showed in Figure 16. The results indicated that these H2L2 antibodies can be divided into two epitope bins. The first group (Bin 1) includes most H2L2 antibodies i.e., PR300147, PR300163, PR300187, PR300193, PR300281, PR300284, and PR300286 which bind to epitope overlapping with the epitope of Amatuximab. The second group (Bin 2) includes PR300162 and PR300283, which bind to an epitope completely different from the epitope of Amatuximab.

Example 7. Antibody Internalization by MSLN Expressing Cells

In this example, pHAb Amine Reactive Dye (Promega, Cat #G9841) was used to determine the antigen-based internalization of anti-MSLN antibodies into COV644 cells. pHAb Dyes are pH sensor dyes that have very low fluorescence at pH > 7 and a dramatic increase in fluorescence when the pH of the solution becomes acidic. When an antibody labelled with pHAb dyes binds outside membrane of cells in neutral pH, no or very low fluorescence could be monitored. After internalization, the fluorescence will become stronger in lower pH environments in endosomes and lysosomes.

Antibodies were labelled with pHAb Dyes and calculated for DARs following the kit instructions. The labelled antibodies were then incubated with COV644 at 4℃ (the internalization activity at this temperature is very low, which was used as background control) or 37℃ for 24 hours. Then a fluorescence with excitation maxima (Ex) at 532nm and emission maxima (Em) at 560nm was detected. The final normalized results are shown as the fluorescence intensity under 37℃subtracting the fluorescence intensity at background under 4℃ and then divided by DARs of pHAb Dye of the antibody. A higher value indicates a higher internalization activity.

The results of internalization rate of anti-MSLN H2L2 and HCAb antibodies are shown in Figure 17. The results indicate that H2L2 clones PR300163, PR300193, and PR300284, as well as HCAb clone PR005548 show good internalization by COV644 cells.

Example 8. Soluble MSLN Interference Assay

Antibodies binding the membrane bound form of MSLN are advantageous for diagnostic and therapeutic purposes. Because they do not bind soluble MSLN, they will have a lower background for imaging applications, and they can be used at a lower dose for therapeutic uses.

The H2L2 antibodies were tested for their bindings to COV644 expressing mesothelin in the presence or absence of 90 nM soluble MSLN (sMSLN) . The experiments were performed follow similar procedure shown in Example 4, except 90 nM soluble MSLN was added to serial diluted antibodies for +sMSLN groups. As shown in the Figures 18-20, the binding of these H2L2 antibodies to COV644 cells all decreased in the presence of 90 nM soluble MSLN as compared to without soluble MSLN. However, some of the antibodies including PR300283 and PR300162, showed less changes, which indicated less interference by soluble MSLN to the binding of these antibodies to membrane-bound form of MSLN.

The HCAb antibodies were also tested for their bindings to membrane-bound form of mesothelin and interference by soluble mesothelin. As shown in Figure 21, 30 nM of the HCAbs were tested by FACS for binding to COV644 cells expressing mesothelin in the presence or absence of 60 nM soluble mesothelin. And as shown in Figure 22, HCAb 19G6 demonstrated binding specificity to the membrane bound-form of mesothelin, in which addition of soluble MSLN did not interfere with 19G6’s binding activity to COV644. Figure 23 showed the method of using Bio-Layer Interferometry (BLI) analysis to test HCAb binding activity to biotin-MSLN bound on streptavidin tips (Octet) in the presence of 60 nM soluble MSLN, in which HCAb 51 8-10 demonstrated binding specificity to the membrane bound-form of mesothelin.

Example 9. Blockade of the MSLN-MUC16 interaction

Antibodies blocking the interaction between MSLN and MUC16 (which plays a role in tumour metastasis) are useful in the treatment of tumours for inhibiting both tumour growth and metastasis. H2L2 and HCAb antibodies were tested for their activity to block the interaction between MSLN and MUC16 expressed on HeLa cells (human epithelial cervix adenocarcinoma line) . In brief, for each sample tested with FACS, 200,000 HeLa cells were mixed with 0.2μg of biotin-tagged soluble MSLN with either 0.5 HCAb (black bars) or 1μg of H2L2 (white bars) . Positive binding was determined using Strep-PE. No antibody was added in the positive control group (C+) , no MSLN was added in the C-2nd group (Strep-PE only control) and neither antibody nor MSLN was added in the C-control group (HeLa only) . The results are shown in Figure 24. “141” and “11” on this figure represent clones 141pl1-4 and 11pl1-4 respectively. The results showed that HCAbs (141pl1-4, 11pl1-4) and H2L2s (3.11A3, 7.5H12) can block MSLN-MUC16 interaction.

Additional HCAb antibodies were also tested for their inhibition on the interaction of soluble MSLN with MUC16 expressed on HeLa cells (Figure 25) . 250,000 HeLa cells expressing MUC16 were seeded per well and the binding of 0.25μg biotinylated MSLN to the cells was measured in the presence of 1μg of HCAb using strep coupled to PE to indicate the MSLN binding with FACS. 2μg of amatuximab was used as a positive control (C+Amatuximab) and an irrelevant antibody was used as a negative control (C-antibody) . The group with no antibody and MSLN added was used to measure the background of the Strep-PE detection (C-2nd) . As shown in Figure 25, HCAbs including 2E12, 11A10, 10E3 can block MSLN-MUC16 interaction.

Furthermore, HCAbs were tested for their capability to block the interaction of mesothelin with MUC16 by bio-layer interferometry (ForteBio Octet) . As shown in Figure 26, biotinylated MSLN was loaded onto the Octet tips followed by binding of the antibody; then the extracellular domain of MUC16 was added (point 0 in the graph) and its binding was recorded over time followed by a washing step, which indicates the stability of the interaction. Top dashed line in the graph is an isotype control. HCAb 11A10 demonstrated stronger MUC16 blocking activity than Amatuximab.

Example 10. ADCC activity determined by reporter assay

The antibodies were tested for ADCC (Figure 27) as any antibody with an active Fc domain would lead to ADCC activity (Kang &Taek (2019) Experimental &Molecular Medicine 51: 1-9) . Jurkat-Lucia^TM NFAT-CD16 cells (InvivoGen) were engineered from the human T-lymphocyte Jurkat cell line, which naturally expresses a functional NFAT pathway. Jurkat-Lucia^TM NFAT-CD16 cells stably express the cell surface Fc receptor CD16A (FcγRIIIA; V158 high affinity allotype) and the Lucia luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements. CHO cells expressing human MSLN (CHOK1-hu MSLN) were mixed with the Jurkat-Lucia^TM NFAT-CD16 cells in the presence of different concentrations of anti-MSLN antibodies, and luciferase activity was determined as a measure of ADCC activity (Figure 27) .

The results showed that, both two different types of antibodies, 19G6 which binds the membrane bound form of MSLN, and 11A10 that can block the interaction between MSLN and MUC16, demonstrated stronger ADCC activity than Amatuximab. ADCC activity would be of added value for therapeutic purposes of MSLN positive tumors.

Example 11. Antibody Engineering for HCAb PR004197

PR004197 has strong binding activities to both human and cyno MSLN expressing cells. The hydrophobicity of PR004197 was determined by Hydrophobic interaction chromatography assay by HPLC (HIC-HPLC) , and it showed delayed retention time (20.5 minutes) which indicated this molecule tends to be hydrophobic in the buffer solution. The thermo-stability of PR004197 was determined by UNCLE instrument (Unchained Labs) , and it showed that its lowest thermo transition temperature (Tm1) was about 56 ℃, as listed in Table 13.

To further improve the biophysical properties of PR004197, variants were made through structure modeling and design, and a few mutations were introduced into framework regions without impeding the antigen binding. In Table 13, two variants (PR006372, PR006373) showed improved hydrophobicity (with faster retention time on HIC-HPLC) and improved thermo-stability (with higher Tm1) comparing to parental HCAb PR004197. Further, the results in Figure 28 and Table 14 showed the two variants (PR006372, PR006373) retained the binding activity to both human and cyno MSLN on cells.

Table 13. Biophysical properties of HCAb PR004197 and its variants

Table 14. Binding activities of HCAb PR004197 and its variants to cell surface MSLN by FACS

Claims

An antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH) , and wherein

(1) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 212, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 226;

(2) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 215, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 226;

(3) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 213, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 227;

(4) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 194, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 220;

(5) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 199, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 220;

(6) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 195, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 221;

(7) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 195, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 225;

(8) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 196, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 222;

(9) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 200, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 222;

(10) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 197, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 223;

(11) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 198, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 224; or

(12) the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 201, and the VL comprises the LCDRs 1-3 of a VL having the amino acid sequence set forth in SEQ ID NO: 224.
The antibody or antigen binding fragment thereof according to claim 1, wherein

(1) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 36, 74, 121 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 149, 161, 177 respectively;

(2) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 37, 75, 122 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 150, 162, 178 respectively;

(3) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 27, 62, 107 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 144, 157, 171 respectively;

(4) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 27, 62, 112 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 144, 157, 171 respectively;

(5) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28, 63, 108 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 145, 158, 172 respectively;

(6) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28, 63, 108 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 145, 158, 176 respectively;

(7) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 29, 64, 109 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 146, 159, 173 respectively;

(8) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 29, 67, 113 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 146, 159, 173 respectively;

(9) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 30, 65, 110 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 147, 160, 175 respectively;

(10) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 31, 66, 111 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 148, 158, 174 respectively; or

(11) the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 31, 66, 114 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 148, 158, 174 respectively,

wherein the CDRs are determined by EU Kabat system.
The antibody or antigen binding fragment thereof according to claim 1 or 2, wherein:

(1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 212, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 226;

(2) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 215, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 226;

(3) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 213, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 227;

(4) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 194, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 220;

(5) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 199, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 220;

(6) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 195, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 221;

(7) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 195, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 225;

(8) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 196, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 222;

(9) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 200, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 222;

(10) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 197, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 223;

(11) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 198, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 224; or

(12) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 201, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 224.
The antibody or antigen binding fragment thereof according to any one of claims 1-3, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) , and wherein:

(1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 259, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 273;

(2) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 262, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 273;

(3) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 260, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 274;

(4) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 238, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 267;

(5) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 245, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 267;

(6) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 240, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 268;

(7) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 240, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 272;

(8) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 241, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 269;

(9) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 246, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 269;

(10) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 243, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 270;

(11) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 244, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 271; or

(12) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 247, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 271.
An antibody that specifically binds to mesothelin, or an antigen binding fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) , and wherein the VH comprises the HCDRs 1-3 of a VH having the amino acid sequence set forth in SEQ ID NO: 209, SEQ ID NO: 214, SEQ ID NO: 204, SEQ ID NO: 203, SEQ ID NO: 185, SEQ ID NO: 211, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 202, SEQ ID NO: 206, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, or SEQ ID NO: 193.
The antibody or antigen binding fragment thereof according to claim 5, wherein the VH comprises HCDRs 1-3 having the amino acid sequences set forth in SEQ ID NOs: 35, 70, 117 respectively, SEQ ID NOs: 38, 76, 123 respectively, SEQ ID NOs: 27, 69, 116 respectively, SEQ ID NOs: 27, 68, 115 respectively, SEQ ID NOs: 22, 56, 100 respectively, SEQ ID NOs: 23, 58 102 respectively, SEQ ID NOs: 39, 77, 124 respectively, SEQ ID NOs: 35, 73, 117 respectively, SEQ ID NOs: 35, 71, 120 respectively, SEQ ID NOs: 24, 58, 102 respectively, SEQ ID NOs: 33, 71, 118 respectively, SEQ ID NOs: 32, 70, 117 respectively, SEQ ID NOs: 34, 72, 119 respectively, SEQ ID NOs: 33, 71, 120 respectively, SEQ ID NOs: 35, 73, 120 respectively, SEQ ID NOs: 23, 57, 101 respectively, SEQ ID NOs: 25, 56, 100 respectively, SEQ ID NOs: 23, 59, 103 respectively, SEQ ID NOs: 23, 60, 104 respectively, SEQ ID NOs: 26, 61, 105 respectively, or SEQ ID NOs: 22, 56, 106 respectively.
The antibody or antigen binding fragment thereof according to claim 5 or 6, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 209, SEQ ID NO: 214, SEQ ID NO: 204, SEQ ID NO: 203, SEQ ID NO: 185, SEQ ID NO: 211, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 202, SEQ ID NO: 206, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, or SEQ ID NO: 193.
The antibody or antigen binding fragment thereof according to any one of claims 5-7, wherein the antibody comprises a heavy chain (HC) , and wherein the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 256, SEQ ID NO: 261, SEQ ID NO: 251, SEQ ID NO: 250, SEQ ID NO: 249, SEQ ID NO: 258, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 248, SEQ ID NO: 253, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 255, , SEQ ID NO: 257, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 275, or SEQ ID NO: 276.
The antibody or antigen binding fragment thereof according to any one of claims 5-8, wherein the antibody does not comprise a light chain.
The antibody or antigen binding fragment thereof according to any one of claims 5-9, wherein the antibody comprises two heavy chains.
The antibody or the antigen binding fragment thereof according to any one of claims 1-10, wherein the antibody or antigen binding fragment thereof specifically binds to membrane bound mesothelin.
The antibody or the antigen binding fragment thereof according to claim 11, wherein the antibody or the antigen binding fragment thereof binds to membrane bound mesothelin with a higher affinity as compared to the affinity of its binding to soluble mesothelin.
The antibody or the antigen binding fragment thereof according to claim 11, wherein the antibody or the antigen binding fragment thereof binds to membrane bound mesothelin with an affinity which is at least two folds, at least three folds, at least five folds, at least 10 folds, at least 20 folds, at least 30 folds, at least 50 folds, or at least 100 folds of the affinity of its binding to soluble mesothelin.
The antibody or the antigen binding fragment thereof according to claim 11, wherein the antibody or the antigen binding fragment thereof does not bind to soluble MSLN.
The antibody or the antigen binding fragment thereof according to any one of claims 1-14, wherein the antibody or the antigen binding fragment thereof blocks the interaction between MSLN and MUC16.
The antibody or antigen binding fragment thereof according to any one of claims 1-15, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.
The antibody or the antigen binding fragment thereof according to any one of claims 1-16, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.
The antibody or the antigen binding fragment thereof according to any one of claims 1-17, wherein the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
The antibody or the antigen binding fragment thereof according to any one of claims 1-18, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab’, F (ab') ₂, Fd, Fd’, Fv, scFv, ds-scFv and dAb.
The antibody or the antigen binding fragment thereof according to any one of claims 1-19, wherein the antibody is a monoclonal antibody, a bi-specific or a multi-specific antibody.
The antibody or the antigen binding fragment thereof according to any one of claims 1-20, wherein the antibody is monovalent, bivalent or multivalent.
The antibody or antigen binding fragment thereof according to any one of claims 1-21, wherein the antibody or antigen binding fragment is attached to a fluorescent label, radiolabel or cytotoxic agent.
The antibody or antigen binding fragment thereof according to any one of claims 1-22, wherein the antibody or antigen binding fragment thereof is obtained using a transgenic non-human animal with human MSLN transgene by the method comprising:

(A) inducing expression of human MSLN in the animal, and

(B) isolating antibodies that specifically bind to MSLN or cells that produce antibody that specifically binds to MSLN;

wherein step (A) is performed after the animal has established central tolerance.
The antibody or antigen binding fragment thereof according to claim 23, wherein the method further comprises:

(A’) reducing or eliminating expression of endogenous MSLN in the animal,

wherein step (A’) is performed preferably before or during the period in which the animal is establishing central tolerance.
The antibody or antigen binding fragment thereof according to claim 23 or cliam 24, wherein the animal is a rodent, optionally a mouse.
The antibody or antigen binding fragment thereof according to claim 25, wherein:

(i) step (A’) is performed before or during the 1-2 weeks after birth of the animal, during the ten days after birth of the animal or for the lifetime of the animal, and/or

(ii) step (A) is performed later than eight days after birth.
The antibody or antigen binding fragment thereof according to any one of claims 1-22, wherein the antibody is obtained using a non-human animal by the method comprising:

(a) reducing or eliminating expression of endogenous MSLN in the animal,

(b) immunising the animal with human MSLN, and

(c) isolating antibodies that specifically bind to MSLN or cells that produce antibody that specifically binds to MSLN;

wherein step (a) is performed before or during the period in which the animal is establishing central tolerance.
The antibody or antigen binding fragment thereof according to claim 27, wherein the animal is a rodent, optionally a mouse.
The antibody or antigen binding fragment thereof according to claim 28, wherein:

(i) step (a) is performed before or during the eight days after birth of the animal, before or during the ten days after birth of the animal or for the lifetime of the animal, and/or

(ii) step (b) is performed later than eight days after birth or later than ten days after birth.
The antibody or antigen binding fragment thereof according to any one of claims 27-29, wherein:

(i) step (a) comprises inducing inactivation of endogenous MSLN expression, and/or

(ii) step (b) comprises inducing expression of hunman MSLN in the animal.
The antibody or antigen binding fragment thereof according to any one of claims 23-27 and 30, wherein the animal comprises:

(i) one or more genes encoding human MSLN and

(ii) one or more genes that encode:

(1) a transactivator that enhances expression of human MSLN, and/or

(2) an inducible nuclease that enhances expression of human MSLN.
The antibody or antigen binding fragment thereof according to claim 31, wherein the inducible nuclease is Cre recombinase (Cre) , flippase (Flp) , a CRISPR associated protein (Cas) or a nuclease that does not cut the DNA of the animal.
The antibody or antigen binding fragment thereof according to claim 31 or claim 32, wherein:

(i) the one or more genes encoding human MSLN are transgenes, and/or

(ii) the one or more genes encoding the transactivator and/or inducible nuclease are transgenes.
The antibody or antigen binding fragment thereof according to claim 33, wherein the transactivator is reverse tetracycline-controlled transactivator (rtTA) , and wherein a Tet Response Element (TRE) is upstream of the gene encoding human MSLN.
The antibody or antigen binding fragment thereof according to any one of claims 23-34, wherein the animal expresses heavy chain-only antibodies.
The antibody or antigen binding fragment thereof according to any one of claims 23-35, wherein the animal expresses tetrameric antibodies comprising two heavy and two light chains.
The antibody or antigen binding fragment thereof according to any one of claims 23-36, wherein the animal comprises one or more transgenic immunoglobulin locus.
The antibody or antigen binding fragment thereof according to claim 37, wherein the one or more transgenic immunoglobulin loci comprise:

(i) one or more heterologous heavy chain loci, each of which comprises at least one V gene segment, at least one D gene segment, at least one J gene segment and at least one constant region; and/or

(ii) one or more heterologous light chain loci, each of which comprises at least one V gene segment, at least one J gene segment and at least one constant region.
The antibody or antigen binding fragment thereof according to claim 37 or claim 38, wherein:

(i) one or more endogenous immunoglobulin heavy chain loci in the animal are deleted or silenced and/or

(ii) one or more endogenous immunoglobulin light chain loci in the animal are deleted or silenced.
A nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof according to any one of claims 1-39.
A vector comprising the nucleic acid according to claim 40.
A host cell comprising the nucleic acid according to claim 40 or the vector according to claim 41.
A pharmaceutical composition comprising (i) the antibody or the antigen binding fragment thereof according to any one of claims 1-39; and (ii) a pharmaceutically acceptable carrier or excipient.
An antibody-drug conjugate (ADC) , comprising the antibody or the antigen binding fragment thereof according to any one of claims 1-39.
A method of treating a cancer in a subject, comprising administering to the subject an effective amount of the antibody or the antigen binding fragment thereof according to any one of claims 1-39, the pharmaceutical composition according to claim 43, or the ADC according to claim 44.
The method according to claim 45, wherein the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer and ovarian cancer.
The method according to claim 45 or 46, further comprising administering to the subject a second therapeutic agent.
The method according to claim 47, wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
A method for diagnosing mesothelin-positive cancer in a subject comprising:

(a) obtaining a biological sample from the subject,

(b) contacting the sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-39, and

(c) detecting binding of the antibody to the sample,

wherein an increase in binding of the antibody or antigen binding fragment thereof to the sample as compared to binding of the antibody or antigen binging fragment thereof to a control sample identifies the subject as having a mesothelin-positive cancer.
A method for imaging a mesothelin-positive cancer in a subject comprising:

(a) administering the antibody or antigen binding fragment thereof according to any one of claims 1-39 to the subject, wherein the antibody is conjugated to a detectable marker, and

(b) detecting the presence of the marker.
The method according to claim 50, wherein:

(a) the detectable marker is ¹¹¹In, and preferably the detection of the marker is by single-photon emission computed tomography, or

(b) the detectable marker is ⁸⁹Zr, and preferably the detection of the marker is by positron emission tomography.
A method of producing an antigen-specific antibody in a transgenic non-human animal comprising:

(A) inducing expression of the antigen in the animal, and

(B) isolating antibodies that specifically bind to the antigen or cells that produce antibody that specifically binds to the antigen;

wherein step (A) is performed after the animal has established central tolerance.
The method according to claim 52, wherein the method further comprises:

(A’) reducing or eliminating expression of a protein that is similar or identical to the antigen in the animal,

wherein step (A’) is performed preferably before or during the period in which the animal is establishing central tolerance.
The method according to claim 52 or claim 53, wherein the animal is a rodent, optionally a mouse.
The method according to claim 54, wherein:

(i) step (A’) is performed before or during the 1-2 weeks after birth of the animal, during the ten days after birth of the animal or for the lifetime of the animal, and/or

(ii) step (A) is performed later than eight days after birth.
A method of producing an antigen-specific antibody in a non-human animal comprising:

(a) reducing or eliminating expression of a protein that is similar or identical to the antigen in the animal,

(b) immunising the animal with the antigen, and

(c) isolating antibodies that specifically bind to the antigen or cells that produce antibody that specifically binds to the antigen;

wherein step (a) is performed before or during the period in which the animal is establishing central tolerance.
The method according to claim 56, wherein the animal is a rodent, optionally a mouse.
The method according to claim 57, wherein:

(i) step (a) is performed before or during the eight days after birth of the animal, before or during the ten days after birth of the animal or for the lifetime of the animal, and/or

(ii) step (b) is performed later than eight days after birth or later than ten days after birth.
The method according to any one of claims 56-58, wherein:

(i) step (a) comprises inducing inactivation of protein expression, and/or

(ii) step (b) comprises inducing expression of the antigen in the animal.
The method according to any one of claims 52-55 and 59, wherein the animal comprises:

(i) one or more genes encoding the antigen and

(ii) one or more genes that encode:

(1) a transactivator that enhances expression of the antigen, and/or

(2) an inducible nuclease that enhances expression of the antigen.
The method according to claim 60, wherein the inducible nuclease is Cre recombinase (Cre) , flippase (Flp) , a CRISPR associated protein (Cas) or a nuclease that does not cut the DNA of the animal.
The method according to claim 60 or claim 61, wherein:

(i) the one or more genes encoding the antigen are transgenes, and/or

(ii) the one or more genes encoding the transactivator and/or inducible nuclease are transgenes.
The method according to claim 62, wherein the transactivator is reverse tetracycline-controlled transactivator (rtTA) , and wherein a Tet Response Element (TRE) is upstream of the gene encoding the antigen.
The method according to any one of claims 52-63, wherein the antigen is:

(i) a protein complex, optionally wherein the antigen is encoded by more than one gene, or

(ii) a posttranslational modification in a protein complex, such as a sugar moiety.
The method according to any one of claims 53-64, wherein:

(i) the protein is an endogenous protein, and/or

(ii) the amino acid sequence of the protein is at least 90%, at least 95%, at least 99%or 100%identical to the amino acid sequence of the antigen.
The method according to any one of claims 53-65, wherein:

(i) the protein is in a first form, and

(ii) expression of the protein in a second form is not reduced or eliminated.
The method according to claim 66, wherein the first and second forms are splice variants.
The method according to claim 66 or claim 67, wherein:

(i) the first form is a membrane-bound form, the second form is a secreted form and the antigen is a protein in membrane-bound form, or

(ii) the first form is a secreted form, the second form is a membrane-bound form and the antigen is a protein in secreted form.
The method according to any one of claims 53-68, wherein:

(i) the protein is an endogenous protein that is essential for development of the animal, and

(ii) the method further comprises introducing a gene that encodes a replacement protein that has a similar function to the endogenous protein but is sufficiently different in amino acid sequence to the antigen to allow immunisation.
The method according to claim 69, wherein:

(i) the amino acid sequence of the replacement protein is less than 80%identical, less than 70%identical, less than 60%identical or less than 50%identical to the amino acid sequence of the antigen, and/or

(ii) the replacement protein is obtained from an animal of a different species.
The method according to claim 69 or claim 70, wherein the gene encoding the replacement protein replaces a gene encoding the endogenous protein.
The method according to any one of claims 52-71, wherein the animal expresses heavy chain-only antibodies.
The method according to any one of claims 52-71, wherein the animal expresses tetrameric antibodies comprising two heavy and two light chains.
The method according to any one of claims 52-73, wherein the animal comprises one or more transgenic immunoglobulin locus.
The method according to claim 74, wherein the one or more transgenic immunoglobulin loci comprise:

(i) one or more heterologous heavy chain loci, each of which comprises at least one V gene segment, at least one D gene segment, at least one J gene segment and at least one constant region; and/or

(ii) one or more heterologous light chain loci, each of which comprises at least one V gene segment, at least one J gene segment and at least one constant region.
The method according to claim 74 or claim 75, wherein:

(i) one or more endogenous immunoglobulin heavy chain loci in the animal are deleted or silenced and/or

(ii) one or more endogenous immunoglobulin light chain loci in the animal are deleted or silenced.
An antigen-specific antibody obtained by the method according to any one of claims 52-76.