WO2018170096A1 - Use of a cd4/cd8 bispecific antibody for the treatment of diabetes - Google Patents

Abstract

The present disclosure is directed to bispecific antibodies binding to CD4 and CD8. In a further embodiment, there is provided a method of treating diabetes in a subject comprising providing the bispecific antibodies binding to CD4 and CD8 to said subject. In some aspects, the diabetes is Type 1 diabetes. A further embodiment provides a kit comprising an antibody or antibody fragment according to the embodiments, e.g., the bispecific antibody or antibody fragment to CD4 and CD8.

Description

DESCRIPTION

USE OF A CD4/CD8 BISPECIFIC ANTIBODY FOR THE TREATMENT OF

DIABETES

PRIORITY CLAIM

This application claims benefit of priority to U. S. Provisional Application Serial No, 62/471 ,227, filed March 14, 2017, the entire contents of which are hereby incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named "DURA_P0004WO_ST25.txt", which is 67 KB (as measured in Microsoft Windows®) and was created on March 14, 2018, is filed herewith by electronic submission and is incorporated by reference herein.

BACKGROUND

1. Field of the Disclosure

The present disclosure relates generally to the fields of biology, medicine, and immunology. More particular, the disclosure relates to bi-specific human antibodies binding to CD4 and CD8.

2. Background

Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period. Symptoms of high blood sugar include frequent urination, increased thirst, and increased hunger. If left untreated, diabetes can cause many complications. Acute complications can include diabetic ketoacidosis, non-ketotic hyperosmolar coma, or death. Serious long-term complications include heart disease, stroke, chronic kidney failure, foot ulcers, and damage to the eyes.

As of 2015, an estimated 415 million people had diabetes worldwide, with type 2 DM making up about 90% of the cases. This represents 8.3% of the adult population, with equal rates in both women and men. As of 2014, trends suggested the rate would continue to rise. Diabetes at least doubles a person's risk of early death. From 2012 to 2015, approximately 1.5 to 5.0 million deaths each year resulted from diabetes. The global economic cost of diabetes in 2014 was estimated to be US$612 billion. In the United States, diabetes cost $245 billion in Diabetes is due to either the pancreas not producing enough insulin or the cells of the body not responding properly to the insulin produced. There are three main types of diabetes mellitus. Type 1 DM results from the pancreas's failure to produce enough insulin. This form was previously referred to as "insulin-dependent diabetes mellitus" (IDDM) or "juvenile diabetes". The cause is unknown.

Prevention and treatment involve maintaining a healthy diet, regular physical exercise, a normal body weight, and avoiding use of tobacco. Control of blood pressure and maintaining proper foot care are important for people with the disease. Type 1 DM must be managed with insulin injections. Type 2 DM may be treated with medications with or without insulin. Insulin and some oral medications can cause low blood sugar. Weight loss surgery in those with obesity is sometimes an effective measure in those with type 2 DM. Gestational diabetes usually resolves after the birth of the baby. However, all of these treatments have drawbacks and limitations, and a more effective treatment is urgently needed, particularly for the treatment of Type 1 DM.

SUMMARY

Thus, in accordance with the present disclosure, antibodies targeted to CD4 and CD8 are provided herein. In a first embodiment, there is provided a bispecific antibody or antibody fragment having binding specificity for CD4 and CD8.

In some aspects, the antibody comprises heavy chain CDRs of SEQ ID NOS: 1-3 and light chain CDRs of SEQ ID NOS: 4-6, and heavy chain CDRs of SEQ ID NOS: 7-9 and light chain CDRs of SEQ ID NOS: 10-12. In certain aspects, the antibody comprises CDR sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR regions of the amino acid sequences of SEQ ID NOS: 1-12.

In certain aspects, the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 14, 16, 18, and 20. In some aspects, antibody or antibody fragment is encoded by heavy and light chain variable sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 14, 16, 18, and 20. In particular aspects, the antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 14, 16, 18, and 20.

In some aspects, the antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 13, 15, 17, and 19. In certain aspects, the antibody or antibody fragment comprises heavy and light chain variable sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 13, 15, 17, and 19. In specific aspects, the antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 13, 15, 17, and 19.

The antibody may be an IgG, a recombinant IgG antibody, or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, a glycan modified antibody with altered (eliminated or enhanced) FcR interactions, such as enzymatic or chemical addition or removal of glycans, a genetically modified glycosylating partem, or an antibody or antibody fragment comprising an Fc portion mutated to enhance FcRn interactions to increase the in vivo half-life and the in vivo effect, such as a YTE or LS mutation. In certain aspects, the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In some aspects, antibody is a chimeric antibody, a human antibody, an IgG antibody or a humanized antibody.

In another embodiment, there is provided a bispecific antibody or antibody fragment having binding specificity for CD4 and CD8, wherein said antibody recognizes a discontinuous epitope found in CD8 residues NEGYYFCSA and/or PRGAAASPTFLLY. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In some aspects, antibody is a chimeric antibody, a human antibody, an IgG antibody or a humanized antibody. The antibody may be an IgG, a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, such as a LALA, N297, GASD/ALIE, a glycan modified antibody with altered (eliminated or enhanced) FcR interactions, such as enzymatic or chemical addition or removal of glycans, a genetically modified glycosylating partem, or an antibody or antibody fragment comprising an Fc portion mutated to enhance FcRn interactions to increase the in vivo half-life and the in vivo effect, such as a YTE or LS mutation.

In a further embodiment, there is provided a method of treating diabetes in a subject comprising providing an antibody or antibody fragment of the embodiments (e.g., the bispecific antibody or antibody fragment to CD4 and CD8) to said subject. In some aspects, the diabetes is Type 1 diabetes. In particular aspects, the subject is a human. In other aspects, the subject is a non-human mammal. In some aspects, the subject is selected from a neonate, a pediatric patient, a teenager, an adult or a patient over about 60 years of age.

In some aspects, the providing is chronic, such as daily, weekly, monthly, every other month, every three months, every four months, every five months, every six months, every nine months or every year. In certain aspects, the effects of providing are persistent.

In certain aspects, providing comprises administering said antibody or antibody fragment to said antibody. In some aspects, providing comprises genetic delivery of an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

In some aspects, providing results in reducing T effector cell number in the subject's pancreas and/or pancreatic lymph nodes, such as CD4⁺ T cells. In certain aspects, providing results in an increase in Fox3+ Treg cell number and/or activity in the subject's pancreas and/or pancreatic lymph nodes.

In certain aspects, the method further comprises administering to said subject a second diabetes therapy. In another embodiment, there is provided a hybridoma or engineered cell encoding an antibody or antibody fragment according to the embodiments (e.g., the bispecific antibody or antibody fragment to CD4 and CD8).

In yet another embodiment, there is provided a vaccine formulation comprising one or more antibodies or antibody fragments according to the embodiments (e.g., the bispecific antibody or antibody fragment to CD4 and CD8).

A further embodiment provides a kit comprising an antibody or antibody fragment according to the embodiments (e.g., the bispecific antibody or antibody fragment to CD4 and CD8).

The term "T cell" refers to T lymphocytes, and includes, but is not limited to, γδ⁺ T cells, NK T cells, CD4+ T cells and CD8+ T cells. CD4+ T cells include THO, THI and T_H2 cells, as well as regulatory T cells (T_reg). There are at least three types of regulatory T cells: CD4+ CD25+ T_reg, CD25 T_H3 T_reg, and CD25 TRI T_reg. "Cytotoxic T cell" refers to a T cell that can kill another cell. The majority of cytotoxic T cells are CD8+ MHC class I-restricted T cells, however some cytotoxic T cells are CD4+. In particular embodiments, the T cell of the present disclosure is CD4+ or CD8+.

The activation state of a T cell defines whether the T cell is "resting" (i. e., in the Go phase of the cell cycle) or "activated" to proliferate after an appropriate stimulus such as the recognition of its specific antigen, or by stimulation with OKT3 antibody, PHA or PMA, etc. The "phenotype" of the T cell (e.g. , naive, central memory, effector memory, lytic effectors, help effectors (THI and TH2 cells), and regulatory effectors), describes the function the cell exerts when activated. A healthy donor has T cells of each of these phenotypes, and which are predominately in the resting state. A naive T cell will proliferate upon activation, and then differentiate into a memory T cell or an effector T cell. It can then assume the resting state again, until it gets activated the next time, to exert its new function and may change its phenotype again. An effector T cell will divide upon activation and antigen-specific effector function.

As used herein, the term "antigen" is a molecule capable of being bound by an antibody or T-cell receptor. An antigen may generally be used to induce a humoral immune response and/or a cellular immune response leading to the production of B and/or T lymphocytes.

"Immune response" broadly refers to the antigen-specific responses of lymphocytes to foreign or self substances. Any substance that can elicit an immune response is said to be "immunogenic" and is referred to as an "immunogen". All immunogens are antigens, however, not all antigens are immunogenic. Immune responses include humoral responses (via antibody activity) and cell-mediated responses (via T cell activation).

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g. , bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g. , the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this disclosure. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins .

A "non-depleting antibody", such as a mAb, is an antibody which depletes fewer than 70%, such as less than 50%, for example from 10% to 25% and, in some aspects, less than 10% of target cells in vivo. They may be used to induce tolerance to a Class I antigen or to a Class II antigen or to an antigen presented by a Class I or Class II antigen. They may be used to induce tolerance to both antigens. In the case of a transplant, for example, Class I and Class II major histocompatibility (MHC) antigens and non-MHC or minor histocompatibility (minors) antigens may be presented. In particular aspects, a non-depleting antibody is used to reduce a population of CD4-positive T cells and/or CD8-positive T cells to less than about 70%, for example less than about 50%, 20% or even 10%, of their normal level. The more difficult it is likely to be to achieve tolerance, the greater the amount of depletion it is desirable to achieve.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the stated number.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-1C -Nondepleting anti-CD4 (YTS177) and -CD8 (YTS105) antibodies induce diabetes remission in NOD mice. (FIG. 1A) Remission was induced in 20/24 (83%) NOD mice and maintained longterm in 19/20 (95%) NOD mice. (FIG. IB) Blood glucose levels were rapidly reduced following YTS177/YTS105 (YTS) treatment. (FIG. 1C) Insulitis was reduced in the islets of long-term remission (>200 days) NOD mice.

FIGS. 2A-2B - Both nondepleting anti-CD4 and -CD8 antibodies are needed for efficient diabetes remission. (FIG. 2A) Anti-CD8 (YTS 105) treated (n=5). (FIG. 2B) Anti-CD4 (YTS177) treated (n=10).

FIGS. 3A-3B - Induction of remission correlates with T cell purging of the pancreas. (FIG. 3 A) 6 days post-YTS177/YTS105 versus isotype control 2A3 antibody treatment (*P<0.05) CD4+ and CD8+ T cell numbers were reduced in the pancreas (Pan) and pancreatic lymph nodes (PLN) but not the spleen of NOD mice. (FIG. 3B) Foxp3+Treg numbers were also reduced in the pancreas and PLN but not the spleen of YTS versus 2 A3 treated NOD mice.

FIGS. 4A-4B - Islet inflammatory milieu in NOD mice is suppressed prior to T cell purging. (FIG. 5A) Schematic of experimental approach. (FIG. 5B) Relative expression of mRNA encoding CD3, proinflammatory cytokines (IL-2, TNFa, IFNy) and chemokines (CXCL9, CXCL10, CXCL13) is reduced in the islets over time in YTS177/YTS105 and 2A3 control treated NOD mice transgenic for the BDC2.5 T cell receptor (NOD.BDC). Results are presented as relative mRNA expression of YTS versus 2A3 cohorts.

FIGS. 5A-5B - CD4 crosslinking of NOD.BDC PLN but not splenic T cells increases sphingosine-1 -phosphate (SIP) chemotaxis. (FIG. 5A) Schematic of experimental approach. (FIG. 5B) Percent migration to S IP in an in vitro transwell assay of CD4+ T cells from the spleen or PLN of NOD.BDC mice treated with YTS177/105 versus control 2A3 (*P<0.05). FIGS. 6A-6B - Proliferation of transferred BDC2.5 CD4⁺ T cells is suppressed in the PLN of long-term remission NOD mice. (FIG. 6A) Schematic of experimental approach. (FIG. 6B) BDC2.5 CD4+ T cells injected into remission versus control NOD mice fail to proliferate in the PLN.

FIGS. 7A-7B - The frequency of Foxp3⁺Treg is increased in the PLN of long- term remission NOD mice (e.g., 200 days post-YTS177/YTS105).

FIGS. 8A-8C - LCMV-specific CD8⁺ T cell reactivity is unaffected by coreceptor therapy. (FIG. 9A) Schematic of the experimental approach. (FIG. 9B) The frequency and (FIG. 9C) number of splenic CD8+ T cells staining with a H2Db tetramer complexed with the LCMV peptide gp33-41.

FIGS. 9A-9C Nondepleting anti-huCD4/CD8a IgG4 protects against xenogeneic (x)GvHD in NRG-huPBL mice. (FIG 9A) Schematic of experimental approach. NRG-huPBL mice were treated with anti-huCD4/CD8a IgG4 and (FIG. 9B) xGVHD monitored based on weight loss (*P<10^-2), and (FIG. 9C) serum IFNy measured 72 hours post-treatment (**P<10^"3).

FIG. 10 - Nondepleting anti-huCD4/CD8a IgG4 has tissue-specific effects on huCD45⁺CD3⁺ CD4⁺ and CD8⁺ T cells in NRG-huPBL mice. NRG-huPBL mice established with 3 healthy donors were treated with anti-huCD4/CD8a IgG4 or control IgG, and CD4+ and CD8+ T cell numbers measured in the pancreas, liver and spleen 72 hours post-treatment.

FIG. 11 - Nondepleting anti-huCD4/CD8a IgG4 selectively affects recently activated tissue-resident T cells. CD69+huCD4+ and CD69+huCD8+ T cells were examined via flow cytometry in NRG-huPBL mice established with a healthy donor, 72 hours post-treatment with anti-huCD4/CD8a IgG4 (solid line), or a control IgG (dashed line); secondary antibody-only used for staining (grey area). FIGS. 12A-12B - Nondepleting bispecific anti-murine CD4/CD8a (HC8.4) binds CD4⁺ and CD8⁺ T cells and induces PLN T cell purging in NOD mice. (FIG. 12A) NOD splenic T cells were treated with the bispecific HC8.4 or left untreated, and HC8.4 binding detected via flow cytometry using an anti-rat IgG2a antibody. (FIG. 12B) NOD mice were treated with YTS177/YTS105, HC8.4 or control 2A3 and T cell (CD3+) numbers in the PLN measured 72 hours post-treatment via flow cytometry. *P<0.01 versus YTS and control 2A3 groups. FIG. 13 - Nondepleting anti-huCD4/CD8a bispecific antibody binding to huCD4+ and huCD8+ T cells in vitro.

FIG. 14 - Nondepleting monospecific anti-huCD4 and -huCD8a in vitro binding to huCD4⁺ and huCD8⁺ T cells respectively.

FIG. 15 - Antibody full length chain nucleotide and amino acid sequences. FIG. 16 - Antibody variable region nucleotide and amino acid sequences.

FIG. 17 - Nondepleting anti-huCD4/CD8a bispecific antibody has increased tissue-specific effects on huCD45⁺CD3⁺ CD4⁺ and CD8⁺ T cells in NRG-huPBL mice over co-therapy of anti-huCD4 + anti-CD8a monospecific antibody cocktail. NRG-huPBL mice were treated with either anti-huCD4/CD8a bispecific antibody, a cocktail of anti-huCD4 + anti-CD8a monospecific antibodies or control IgG, and CD4+ and CD8+ T cell numbers measured in the pancreas, liver and spleen 72 hours post-treatment.

FIGS. 18-20 - (RESERVED)

FIGS. 21A-B - (FIG. 21A) Human CD4 Protein sequence (only the extracellular domain was used for array design. (FIG. 2 IB) Rendering of the CD4 homodimer as present in lcdh.pdb.

FIG. 22 - The target linear sequence is converted into a library of all overlapping linear peptides directly synthesized on a proprietary solid support called "mini- card". Binding of antibodies is quantified using an automated ELISA-type readout. Constructs containing right amino acid sequence in the correct conformation best bind the antibody.

FIG. 23 - Using CLIPS technology, peptides derived from native proteins are transformed into CLIPS constructs with a range of structures. From left to right: single mP2 loop, stabilized beta sheet, alpha helix, T3 double loop.

FIG. 24 - The target protein contains a-helixes, β-sheets separated by loops is converted into different conformational libraries using a CLIPS scaffold. In this example only single loop mimics are shown. Peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs (right). Binding of antibodies is quantified using an automated ELISA- type read-out. Constructs containing the right amino acid sequence in the correct conformation best bind the antibody.

FIG. 25 - Box plot schematic. Source: flowingdata.com FIG. 26 - Intensity profile and corresponding heatmap representation of a response from a polyclonal serum screened on a library of overlapping linear peptides.

FIG. 27 - Box plot graphs of raw data of antibody screening. The bottom and top of the boxes are the 25th and 75th percentile of the data. The band near the middle of the box is the 50th percentile (the median). The whiskers are at 1.5 the inter- quantile range, an indication of statistical outliers within the dataset (McGill et al, (1978) The American Statistician, 32: 12-16).

FIG. 28 - Overlay of intensity profiles recoded for CD4 mAb under low stringency conditions with linear peptides (solid line) and double Ala analogs (dashed line). Signal intensities are plotted on the y axis and positions of the last residues of a peptide with respect to the target sequence is on the x axis.

FIG. 29 - Overlay of intensity profiles recoded for CD4 mAb under low stringency conditions with linear and conformational epitope mimics. Signal intensities recorded with linear epitope mimics are colored in blue, with looped epitope mimics - in purple, with a-helical epitope mimics - in green, and with β- turn epitope mimics in red. Signal intensities are plotted on the y axis and positions of the last residues of a peptide with respect to the target sequence is on the x axis. FIG. 30 - Heatmap representation of the intensity profile recorded for CD4 mAb under low stringency conditions with disulfide bridge epitope mimics. Please note that only existing S-S combinations have been made. Non-existing S-S combinations are shown in white. High signals are plotted in red and background signals are plotted in black.

FIGS. 31A-B - (FIG. 31 A) Human CD8 Protein sequence (only the extracellular domain was used for array design. (FIG. 2 IB) Rendering of the CD 8 homodimer as present in lcdh.pdb.

FIG. 32. Intensity profile and corresponding heatmap representation of a response from a polyclonal serum screened on a library of overlapping linear peptides. FIG. 33. Box plot graphs of raw data of antibody screening. The bottom and top of the boxes are the 25th and 75th percentile of the data. The band near the middle of the box is the 50th percentile (the median). The whiskers are at 1.5 the inter- quantile range, an indication of statistical outliers within the dataset (McGill et al, (1978) The American Statistician, 32: 12-16). FIG. 34. Overlay of intensity profiles recorded for antibody CD8 mAb under low stringency conditions with linear (blue), single loop (purple), a-helical (green) and β-turn (red) epitope mimics. Signal intensities are plotted on the y axis. Position of the last residue of a peptide with respect to the target sequence is on the x axis. FIG. 35. Heatmap representation of the intensity profile recorded for antibody CD8 mAb under low stringency conditions with disulfide bridge mimics. Note, only natural S-S combinations were made (as per UniProt). Non-existing S-S combinations are mapped in white. High signals are plotted in red and background signals are plotted in black.

FIG. 36. Cartoon rendering of the CD8 dimer based on lakj.pdb. One CD8 unit is rendered in white and the other one in grey colors. Peptide stretches 60 PRGAAASPTFLLY 72 and 109 NEGYYFCSA 117 are shown in blue and red, respectively.

FIG. 37. Molecular surface of the CD8 dimer with single subunits colored in white and grey. Peptide stretches 60 PRGAAASPTFLLY 72 and 109 NEGYYFCSA17 are shown in blue and red, respectively.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, there remains a great need for improved therapies for diabetes, particularly Type I Diabetes. There is much evidence to suggest that CD8⁺ T cells play a role in the development of diabetes (Phillips et al, Rev Diabet Stud., 6(2):97-103, 2009). Early studies in NOD mice showed that the transfer of diabetes by spleen cells from diabetic donors into immuno-compromised recipients required the presence of both CD4⁺ and CD8⁺ T cells (Hutchings et al, JAutoimmun., 3: 1750185, 1990). Further studies in NOD mice showed that CD8⁺ T cells were required for cyclophosphamide-induced diabetes and also that MHC class I expression was required in NOD mice for diabetes to spontaneously develop. Depletion of CD8⁺ T cells has been shown to afford protection from disease and an overall reduction in islet infiltration (Wang et al. Eur J Immunol. 26: 1762-1769, 1996), which has led to the proposition that CD8⁺ T cells may facilitate recruitment of lymphocytes to the pancreas. This interpretation is supported by studies which showed that early administration of an IgG2a rat anti-mouse CD8 monoclonal antibody prevents insulitis in NOD mice, with no effect of such treatment at later time points. However, the requirement for CD8⁺ T cells in the development of diabetes in NOD mice was not a universal observation. It was suggested that CD4⁺ T cells alone could transfer diabetes to NOD.scid recipients, if they were derived from a diabetic donor, but that CD8⁺ T cells were required if the CD4 population was derived from pre-diabetic mice (Christianson ei a/., Diabetes, 442:44-45, 1993).

Every in vivo antibody study targeting CD8⁺ T cells has used antibodies directed at

CD8a. The antibodies used to deplete CD8⁺ T cells in vitro have also been directed against the CD8a chain. There are several cell types apart from αβ T cells that express CD8a including γδ T cells, NKT cells, and some dendritic cells (DCs). In these cases the αα homodimer is expressed. This means that all previous studies could not distinguish between effects on αβ T cells and on other cell types. While it is clear the both CD4⁺ T cells and CD8⁺ T cells play some role in the development of diabetes, there remains a need for effective methods for depleting both cell populations.

Accordingly, CD4/CD8 bispecific antibodies are provided herein which have been demonstrated to provide an effective treatment for diabetes. These and other aspects of the disclosure are described in detail below. I. Diabetes

In some embodiments, the antibodies and antibody fragments provided herein are used for the treatment of diabetes, such as Type 1 diabetes (T1D), or diabetes mellitus type 1, which is a form of diabetes mellitus. Type 1 diabetes is an autoimmune disease that results in the permanent destruction of insulin-producing β cells of the pancreas. Type 1 is lethal unless treatment with exogenous insulin via injections replaces the missing hormone, or a functional replacement for the destroyed pancreatic beta cells is provided (such as via a pancreas transplant).

Type 1 diabetes (formerly known as "childhood," "juvenile" or "insulin-dependent" diabetes) is not exclusively a childhood problem. The adult incidence of type 1 is noteworthy - many adults who contract type 1 diabetes are misdiagnosed with type 2 due to the misconception of type 1 as a disease of children - and since there is no cure, all children with type 1 diabetes will grow up to be adults with type 1 diabetes.

There is currently no preventive measure that can be taken against type 1 diabetes. Most people affected by type 1 diabetes are otherwise healthy and of a healthy weight when onset occurs, but they can lose weight quickly and dangerously, if not diagnosed in a relatively short amount of time. Diet and exercise cannot reverse or prevent type 1 diabetes. Although there are clinical trials ongoing that aim to find methods of preventing or slowing its development, so far none have proven successful, at least on a permanent basis.

The most useful laboratory test to distinguish type 1 from type 2 diabetes is the C- peptide assay, which is a measure of endogenous insulin production since external insulin (to date) has included no C-peptide. However, C-peptide is not absent in type 1 diabetes until insulin production has fully ceased, which may take months. The presence of anti-islet antibodies (to Glutamic Acid Decarboxylase, Insulinoma Associated Peptide-2 or insulin), or lack of insulin resistance, determined by a glucose tolerance test, would also be suggestive of type 1. As opposed to that, many type 2 diabetics still produce insulin internally, and all have some degree of insulin resistance. Testing for GAD 65 antibodies has been proposed as an improved test for differentiating between type 1 and type 2 diabetes.

The cause of type 1 diabetes is still not fully understood. Some theorize that type 1 diabetes could be a virally-induced autoimmune response. Autoimmunity is a condition where one's own immune system "attacks" structures in one's own body either destroying the tissue or decreasing its functionality. In the proposed scenario, pancreatic beta cells in the Islets of Langerhans are destroyed or damaged sufficiently to abolish endogenous insulin production. This etiology makes type 1 distinct from type 2 diabetes mellitus. It should also be noted that the use of insulin in a patient's diabetes treatment protocol does not render them as having type 1 diabetes, the type of diabetes a patient has is determined only by disease etiology. The autoimmune attack may be triggered by reaction to an infection, for example by one of the viruses of the Coxsackie virus family or German measles, although the evidence is inconclusive.

This vulnerability is not shared by everyone, for not everyone infected by these organisms develops type 1 diabetes. This has suggested a genetic vulnerability and there is indeed an observed inherited tendency to develop type 1. It has been traced to particular HLA genotypes, though the connection between them and the triggering of an auto-immune reaction is poorly understood. Wide-scale genetic studies have shown links between genetic vulnerabilities for type 1 diabetes and Multiple Sclerosis and Crohn's Disease.

Some researchers believe that the autoimmune response is influenced by antibodies against cow's milk proteins. A large retrospective controlled study published in 2006 strongly suggests that infants who were never breastfed had a risk for developing type 1 diabetes twice that of infants who were breastfed for at least three months. The mechanism, if any, is not understood. No connection has been established between autoantibodies, antibodies to cow's milk proteins, and type 1 diabetes. A subtype of type 1 (identifiable by the presence of antibodies against .beta, cells) typically develops slowly and so is often confused with type 2 diabetes (see below). In addition, a small proportion of type 1 cases have the hereditary condition maturity onset diabetes of the young (MODY; see below) which can also be confused with type 2.

Vitamin D in doses of 2000 IU per day given during the first year of a child's life has been connected in one study in Northern Finland (where intrinsic production of Vitamin D is low due to low natural light levels) with an 80% reduction in the risk of getting type 1 diabetes later in life. Some suggest that deficiency of Vitamin D3 (one of several related chemicals with Vitamin D activity) may be an important pathogenic factor in type 1 diabetes independent of geographical latitude.

Some chemicals and drugs specifically destroy pancreatic cells. Vacor (N-3- pyridylmethyl-N'-p-nitrophenyl urea), a rodenticide introduced in the United States in 1976, selectively destroys pancreatic β cells, resulting in type 1 diabetes after accidental or intentional ingestion. Vacor was withdrawn from the U. S. market in 1979. Zanosar® is the trade name for streptozotocin, an antibiotic and antineoplastic agent used in chemotherapy for pancreatic cancer that kills β cells, resulting in loss of insulin production. Other pancreatic problems, including trauma, pancreatitis or tumors (either malignant or benign), can also lead to loss of insulin production. The exact cause(s) of type 1 diabetes are not yet fully understood, and research on those mentioned, and others, continues.

Untreated type 1 diabetes can lead to one form of diabetic coma, diabetic ketoacidosis, which can be fatal. Other aspects of the disease include excess gluconeogenesis, excess glycogenolysis, hyperglycemia, hyperglucagonemia, ketosis, hypertriglyceridemia, elevated plasma free fatty acid, weight loss, hypertension, diabetic nephropathy, renal insufficiency, renal failure, hyperphagia, muscle wasting, diabetic neuropathy, and diabetic retinopathy.

Type 1 is treated with insulin replacement therapy - usually by injection or insulin pump, along with attention to dietary management, typically including carbohydrate tracking, and careful monitoring of blood glucose levels using glucose meters. At present, insulin treatment must be continued for a lifetime; this will change if better treatment, or a cure, is discovered. Continuous glucose monitors have been developed which can alert patients to the presence of dangerously high or low blood sugar levels, but the lack of widespread insurance coverage has limited the impact these devices have had on clinical practice so far.

There are both short- and long-term disadvantages to insulin therapy. The main short- term issue concern with insulin monotherapy is the instability of the daily glucose profiles achieved by peripheral injections of insulin. Even optimally controlled patients with at target HgbAlc values have daily spikes of hyperglycemia, with occasional hypoglycemic dips. This may be the result of the enormous anatomical disadvantage of peripherally injected insulin, which cannot meet the high insulin requirements of proximal targets such as alpha cells and hepatocytes without far exceeding the insulin requirements of distal targets such as muscle and fat. The intra-islet concentration of endogenous insulin that perfuses alpha cells in normal islets has been estimated to be over 20 times higher than the levels generated by peripheral injection, and the concentration of endogenous insulin perfusing the liver is 4- to 5-times higher. This means that even a high concentration of exogenous insulin in peripheral plasma may not approach the physiologic levels of endogenous insulin that perfuse these two proximal insulin targets, which control endogenous glucose production.

A second important disadvantage of injected insulin is its inability to respond on a minute-to-minute basis to changes in need, in particular, to lower it instantly when glucose levels are falling. These facts suggest that, if the wild and inappropriate oscillations of insulin and glucagon that create inappropriate swings in hepatic glucose production could be chronically stabilized by suppressing glucagon independently of insulin, the total daily dose of insulin could be reduced to levels that would manage postprandial hyperglycemia and nothing else. The hope is that this would establish a more stable pattern of glucoregulation.

A third important contributing factor to glucose lability is lipolytic lability, which intermittently floods the target tissues of insulin with fatty acids that impair sensitivity to insulin action on glucose metabolism. This contributes to instability of glucose levels, which can fluctuate from dangerously low levels of hypoglycemia to undesirably high hyperglycemia, making frequent blood glucose determination and multiple insulin injections mandatory, thereby significantly lowering the quality of life for patients.

The major long-term effect of insulin therapy is insulin resistance, a well characterized component of type 1 diabetes. As in T2DM, the degree of insulin resistance is closely associated with risk of cardiovascular disease. The high prevalence of coronary artery disease among patients with T1DM is traditionally ascribed to the disease rather than to life-long insulin monotherapy. The role of insulin in the macrovascular complications of T1DM deserves to be examined more closely, given the relationship between the diet-driven endogenous hyperinsulinemia of obesity and the metabolic syndrome, particularly in insulin-resistant patients treated with U-500 insulin. Insulin is a powerful lipogenic force; a life-time of exogenous hyperinsulinemia in T1DM could also cause a form of metabolic syndrome, with insulin resistance, hyperlipidemia, hypercholesterolemia, coronary artery disease and lipotoxic cardiomyopathy and occasionally obesity.

In more extreme cases, a pancreas transplant can help restore proper glucose regulation. However, the surgery and accompanying immunosuppression required is considered by many physicians to be more dangerous than continued insulin replacement therapy and is therefore often used only as a last resort (such as when a kidney must also be transplanted or in cases where the patient's blood glucose levels are extremely volatile). Experimental replacement of β cells (by transplant or from stem cells) is being investigated in several research programs and may become clinically available in the future. Thus far, β cell replacement has only been performed on patients over age 18, and with tantalizing successes amidst nearly universal failure.

Pancreas transplants are generally recommended if a kidney transplant is also necessary. The reason for this is that introducing a new kidney requires taking immunosuppressive drugs anyway, and this allows the introduction of a new, functioning pancreas to a patient with diabetes without any additional immunosuppressive therapy. However, pancreas transplants alone can be wise in patients with extremely labile type 1 diabetes mellitus. Less invasive than a pancreas transplant, islet cell transplantation is currently the most highly used approach in humans to temporarily cure type 1 diabetes. In one variant of this procedure, islet cells are injected into the patient's liver, where they take up residence and begin to produce insulin. The liver is expected to be the most reasonable choice because it is more accessible than the pancreas, and the islet cells seem to produce insulin well in that environment. The patient's body, however, will treat the new cells just as it would any other introduction of foreign tissue. The immune system will attack the cells as it would a bacterial infection or a skin graft. Thus, the patient also needs to undergo treatment involving immunosuppressants, which reduce immune system activity.

Recent studies have shown that islet cell transplants have progressed to the point that

58% of the patients in one study were insulin independent one year after the operation. Ideally, it would be best to use islet cells which will not provoke this immune reaction, but investigators are also looking into placing islets into a protective coating which enables insulin to flow out while protecting the islets from white blood cells.

II. CD4 and CD8

A. Non-Depleting Bispecific Anti-CD4 and Anti-CD8 Antibody

In some embodiments, an antibody as disclosed herein is targeted to CD4 and CD8 antigens. In particular embodiments, the antibody is a bispecific antibody targeted to both CD4 and CD8. In specific embodiments, the bispecific antibody provided herein is a non-depleting antibody which binds to CD4 and CD8 as a receptor antagonist to decrease CD4⁺ T cell or CD8⁺ T cell activation or proliferation without causing lysis or cell destruction.

B. CD4

Cluster of differentiation 4 (CD4) is a glycoprotein having a molecular weight of about

55 kDa, which is expressed on the cell surface of most thymic cells, peripheral blood T cells, monocytes, and macrophages. CD4 is a co-receptor that assists the T cell receptor (TCR) in communicating with an antigen-presenting cell. Using its intracellular domain, CD4 amplifies the signal generated by the TCR by recruiting an enzyme, the tyrosine kinase Lck, which is essential for activating many molecular components of the signaling cascade of an activated T cell.

CD4 is a type I transmembrane protein in which four immunoglobulin superfamily domains (designated in order as D l to D4 from the N terminal to the cell membrane side) are present on the outside of the cells, and two N-linked sugar chains in total are bound to the domains D3 to D4. CD4 binds to a major histocompatibility complex (MHC) class II molecule through Dl and D2 domains, and then activates the T cells. Further, it is also known that CD4 polymerizes through D3 and D4 domains. The Dl domain of CD4 is known to serve as a receptor for a human immunodeficiency virus (HIV) (Anderson et al, Clinical Immunology and Immunopathology, 84(l):73-84), 1997).

Examples of CD4-positive cells include CD4-positive T cells such as a Thl cell, a Th2 cell, a Thl7 cell, a regulatory T cell (Treg), and a γδΤ cell. Further, CD4-positive cells are associated with diseases including cancer and inflammatory diseases (e.g., autoimmune disease or an allergic disease).

The anti-CD4 monoclonal antibody OKT4 was first confirmed as a monoclonal antibody which binds to CD4. Since then, a large number of monoclonal antibodies against CD4 have been reported, most of which are known to recognize the Dl domain. Various anti- CD4 mAbs are under clinical development for the purpose of treating cancers, immune diseases, and infections. For example, based on the fact that the binding between CD4 and HIV is essential for the infection of HIV, an antibody which recognizes Dl domain of CD4 can inhibit the infection of HIV, under the development as an HIV therapeutic agent.

Examples of anti-CD4 mAbs developed as a therapeutic agent for cancers or immune diseases include zanolimumab (6G5) and keliximab (CE9.1). These antibodies are antibodies which exert their medicinal efficacy by specifically attacking CD4-expressing cells which are target cells, and it is considered that the mechanism of medicinal efficacy is mainly due to an ADCC activity (Kim er a/., Blood, 109(11):4655-4662, 2007).

C. CD8

CD8 is a surface glycoprotein that functions as a co-receptor for TCR recognition of peptide antigen complexed with MHC Class I molecule (pMHCI). It is expressed either as an αα homodimer or as an αβ heterodimer (Zamoyska, Immunity, 1 :243-6, 1994), both chains expressing a single extracellular Ig superfamily (IgSF) V domain, a membrane proximal hinge region, a transmembrane domain, and a cytoplasmic tail. CD8 interacts with im and the a2 and a3 domains of MHC Class I molecules using its β strands and the complementary determining regions (CDRs) within the extracellular IgSF V domain. This association increases the adhesion/avidity of the T cell receptor with its Class I target.

In addition, an internal signaling cascade mediated by the CD8a chain associated tyrosine protein kinase p561ck⁴'⁵ leads to T cell activation. Lck is required for activation and expansion of naive CD8+ T cells; however its expression is not essential for responses of memory CD8⁺ T cells to secondary antigenic stimulation in vivo or in vitro (Bachman et al, J Exp Med, 189: 1521-30, 1999). As shown by either CD8a or CD8B gene targeted mice, CD8 plays an important role in the maturation and function of MHC Class I-restricted T lymphocytes (Nakayama et al, Science, 263: 1131-3, 1984). One patient suffering from repeated bacterial infections was found to display a CD8 deficiency due to a single mutation in the CD8a gene. The lack of CD8 did not appear to be essential for either CD8⁺ T cell lineage commitment or peripheral cytolytic function (de la Calle-Martin et al, J Clin Invest, 108: 117- 23, 2001).

The human CD8 molecule is a glycoprotein and cell surface marker expressed on cytotoxic T-cells (CTLs). These are a subset of T-lymphocytes and play an important role in the adaptive immune system of vertebrates. They are responsible for the elimination of virus- infected cells or other abnormal cells such as some tumor cells. These cells are specifically recognized via the T-cell receptor (TCR), which interacts with the certain antigen presented via MHC (major histocompatibility complex) class I on target cells.

There are several anti-CD8 antibodies, including monoclonal antibodies, known in the art, including: 2D2; 4D12.1; 7B12 IG1 1 ; 8E-1.7; 8G5; 14; 21Thy; 51.1 ; 66.2; 109-2D4; 138- 17; 143-44; 278F24; 302F27; AICD8.1 ; anti-T8; B9.1.1; B9.2.4; B9.3.1 ; B9.4.1 ; B9.7.6; B9.8.6; B9.1 1; B9.1 1.10; BE48; BL15; BL-TS8; BMAC8; BU88; BW135/80; C1-11G3; CIO; C12/D3; CD8-4C9; CLB-T8/1 ; CTAG-CD8, 3B5; F80-1D4D11 ; F101-87 (S-T8a); GIO-I; GlO-1.1; HI208; HI209; HI212; HIT8a; HIT8b; HIT8d; ICO-31 ; ICO-122; IP48; ITI-5C2; ITM8-1; JML-H7; JML-H8; L2; L533; Leu-2a; LT8; LY17.2E7; LY19.3B2; M236; M-T122; M-T415; M-T805; M-T806; M- T807; M-T808; M-T809; M-T1014; MCD8; MEM-31; MEM- 146; NU-Ts/c; OKT8; OKT8f; P218; RPA-T8; SM4; T8; T8 /2T8-19; T8 /2T8-2A1 ; T8 /2T8- 1B5; T8 /2T8-1C1 ; T8 /7Pt3F9; T8 /21thy2D3; T8 /21 thy; T8 /TPE3FP; T8b; T41D8; T811 ; TU68; TU102; UCHT4; VIT8; VIT8b; WuT8-l; X107; YTC141.1; and/or YTC 182.20.

D. Non-Depleting Antibodies

U.S. Patent No. 6056956 discloses CD4 mAbs shown to create a tolerance-permissive environment in vivo with which can be achieved tolerance to certain soluble protein antigens as well as transplantation antigens. However, the mechanism(s) by which CD4 mAbs produce these effects are still not clear. In most previous reports, immunosuppression was obtained under conditions that depleted target cells in vivo. A simple interpretation was that the immune suppression so achieved was due to the absence of CD4⁺ T cells. On the other hand, in vitro work has demonstrated that CD4 (and CD8) mAbs could affect lymphocyte functions simply through binding to the antigen on the cell surface without cell lysis. In addition, immunosuppression and tolerance induction has been obtained in with the use of sublytic concentrations of CD4 mAbs and by F(ab')2 CD4 mAb fragments which suggest that for mAb-mediated immune regulation, depletion of target cells may not be essential.

Previous studies have used antibodies that deplete CD4 cells. It has also been found that non-depleting CD4 and CD8 antibodies can also produce tolerance to foreign immunoglobulins, bone marrow and skin grafts. There are advantages of using non-depleting mAbs in vivo. For example, injection of a short course of non-depleting mAbs allows quicker recovery of competent cells from blockade and therefore may lessen the risk of opportunistic infection and other complications due to immune deficiency (e.g., leukemia relapse after T- depleted bone-marrow transplantation) following treatment. III. Monoclonal Antibodies and Production Thereof

A. General Methods

It will be understood that monoclonal antibodies binding to CD4 and CD8 will have several therapeutic applications. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g. , Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265).

The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection. As is well known in the art, a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of nonspecific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

In the case of human antibodies against natural pathogens, a suitable approach is to identify subjects that have been exposed to the pathogens, such as those who have been diagnosed as having contracted the disease, or those who have been vaccinated to generate protective immunity against the pathogen. Circulating anti-pathogen antibodies can be detected, and antibody producing B cells from the antibody -positive subject may then be obtained.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^"6 to 1 x 10^{" 8}. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary particular preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. Ouabain is added if the B cell source is an Epstein Barr virus (EBV) transformed human B cell line, in order to eliminate EBV transformed lines that have not fused to the myeloma.

The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g. , hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain may also be used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.

Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g. , a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supematant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.

MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used to generate monoclonals. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 10⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes antibody-therapeutic agent conjugates. B. Antibodies of the Present Disclosure

The antibodies according to the present disclosure may be defined, in the first instance, by binding specificity. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims. In the present application, particular binding specificities are for CD4 and CD8.

In another aspect, there are provided monoclonal antibodies having CDRs from the heavy and light chains as illustrated herein. Such antibodies may be produced by the clones discussed below.

In yet another aspect, the antibodies may be defined by their variable sequence, which include additional "framework" regions. Furthermore, the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C, (e) the amino acids may vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) the amino acids may vary from those set out above by permitting conservative substitutions (discussed below).

Finally, antibodies may be defined by the activities associated herewith. The present antibodies are characterized as (a) non-depleting, (b) having the ability to reduce effect T cell number in the pancreas or pancreatic lymph nodes, such as CD4+ T cells, and (c) having the ability to increase Fox3+ Treg activity and/or number in the pancreas or pancreatic lymph nodes. C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. The following is a general discussion of relevant techniques for antibody engineering. Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.

The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.

Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. 5,168,062 , U.S. 4,510,245 and U.S. 4,968,615. The recombinant expression vectors can also include origins of replication and selectable markers (see, e.g. , U.S. 4,399,216 , 4,634,665 and). Suitable selectable markers include genes that confer resistance to drugs such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate and the neo gene confers resistance to G418.

Transfection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, calcium-phosphate precipitation, and DEAE-dextran transfection.

Suitable mammalian host cells for expressing the antibodies, antigen binding portions, or derivatives thereof provided herein include Chinese Hamster Ovary (CHO cells), including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216- 4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621, NSO myeloma cells, COS cells and SP2 cells. In some embodiments, the expression vector is designed such that the expressed protein is secreted into the culture medium in which the host cells are grown. The antibodies, antigen binding portions, or derivatives thereof can be recovered from the culture medium using standard protein purification methods.

Antibodies of the disclosure or an antigen-binding fragment thereof can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to ammonium sulfate or ethanol precipitation, acid extraction, Protein A chromatography, Protein G chromatography, anion or cation exchange chromatography, phospho-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.

Antibodies of the present disclosure or antigen-binding fragment thereof include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present disclosure can be glycosylated or can be non- glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20. Therefore an object of the present disclosure are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.

Another object of the present disclosure is a method of using the host cell to produce an antibody and antigen binding fragments, comprising culturing the host cell under suitable conditions and recovering said antibody. Therefore, another object of the present disclosure is the antibody as described in the present disclosure produced with the host cells of the present disclosure and purified to at least 95% homogeneity by weight. Antibody molecules will comprise fragments (such as F(ab'), F(ab')₂) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.

In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g. , a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgGi can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency. Modifications in the Fc region can be introduced to extend the in vivo half-life of the antibody, or to alter Fc mediated fucntions such as complement activation, antibody dependent cellular cytotoxicity (ADCC), and FcR mediated phagocytosis.

Other types of modifications include residue modification designed to reduce oxidation, aggregation, deamidation, and immunogenicity in humans. Other changes can lead to an increase in manufacturability or yield, or reduced tissue cross-reactivity in humans.

Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.

D. Single Chain Antibodies

A Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains. Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alaine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 x 10⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.

The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i. e. , the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).

Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g. , a stablizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero- bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g. , N-hydroxy succinimide) and the other reacting with a thiol group (e.g. , pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g. , the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g. , the selective agent).

It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.

The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g. , the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-l,3'-dithiopropionate. The N-hydroxy- succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

U.S. Patent 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug. U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.

E. Engineering of Bispecific Antibodies

A wild-type IgG antibody contains two identical fragments termed "fragment, antigen binding" (or Fab), each of which is composed of the VH and CHI domains of one heavy chain and the VL and CL domains of a light chain. Each Fab directs binding of the antibody to the same antigen. As used herein, the term "bi-specific antibody" or "IgG BsAb" refers to an IgG antibody comprising two distinct Fabs, each of which direct binding to a separate antigen, and composed of two distinct heavy chains and two distinct light chains. The VH and CHI domains of one heavy chain associate with the VL and CL domains of one light chain to form a "first" Fab, whereas the VH and CHI domains of the other heavy chain associate with the VL and CL domains of the other light chain to form a "second" Fab. More particularly, the term "bi-specific antibody", as used herein, refers to an IgGl, IgG2 or IgG4 class of bi-specific antibody. Even more particular, the term "bi-specific antibody" refers to an IgGl or IgG4 class of bi-specific antibody, and most particularly an IgGl class.

The methods exemplified herein can be used to co-express two distinct Fab moieties, for example Fabs with different Fv regions, with reduced mismatching of respective HC/LC pairs. In addition to bi-specific antibodies and individual Fabs, the methods of the present disclosure can be employed in the preparation of other bi-specific antigen binding compounds. As used herein, the term "bi-specific antigen binding compound" refers to Fab-Fab and IgG- Fab molecules.

The methods and antibodies of the present disclosure comprise designed amino acid modifications at particular residues within the variable and constant domains of heavy chain and light chain polypeptides. As one of ordinary skill in the art will appreciate, various numbering conventions may be employed for designating particular amino acid residues within IgG variable region sequences. Commonly used numbering conventions include Kabat and EU index numbering (see, Kabat et al , Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Other conventions that include corrections or alternate numbering systems for variable domains include Chothia (Chothia C, Lesk A M (1987), J Mol Biol 196: 901-917; Chothia, et al. (1989), Nature 342: 877-883), IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27: 55-77), and AHo (Honegger A, Pluckthun A (2001) J Mol Biol 309: 657-670). These references provide amino acid sequence numbering schemes for immunoglobulin variable regions that define the location of variable region amino acid residues of antibody sequences. Unless otherwise expressly stated herein, all references to immunoglobulin heavy chain variable region (i.e., VH) amino acid residues (i.e., numbers) appearing in the Examples and Claims are based on the Kabat numbering system, as are all references to VL and CL residues. All references to immunoglobulin heavy chain constant region CHI and hinge appearing in the Examples and Claims are also based on the Kabat system, whereas all references to immunoglobulin heavy chain constant regions CH2, and CH3 are based on the EU Index numbering system. With knowledge of the residue number according to Kabat or EU Index numbering, one of ordinary skill can apply the teachings of the art to identify amino acid sequence modifications within the present disclosure, according to any commonly used numbering convention. Note, while the Examples and Claims of the present disclosure employ Kabat or EU Index to identify particular amino acid residues, it is understood that the SEQ IDs appearing in the Sequence Listing accompanying the present application, as generated by Patent In Version 3.5, provide sequential numbering of amino acids within a given polypeptide and, thus, do not conform to the corresponding amino acid numbers as provided by Kabat or EU index.

However, as one of skill in the art will also appreciate, CDR sequence length may vary between individual IgG molecules and, further, the numbering of individual residues within a CDR may vary depending on the numbering convention applied. Thus, to reduce ambiguity in the designation of amino acid residues within CDRs, the disclosure of the present disclosure first employs Kabat to identify the N-terminal (first) amino acid of the HFR3. The amino acid residue to be modified is then designated as being four (4) amino acid residues upstream (i.e. , in the N-terminal direction) from the first amino acid in the reference HFR3. For example, Design A of the present disclosure comprises the replacement of a WT amino acid in HCDR2 with a glutamic acid (E). This replacement is made at the residue located four amino acids upstream of the first amino acid of HFR3, according to Kabat. In the Kabat numbering system, amino acid residue X66 is the most N-terminal (first) amino acid residue of variable region heavy chain framework three (HFR3). One of ordinary skill can employ such a strategy to identify the first amino acid residue (most N-terminal) of heavy chain framework three (HFR3) from any human IgGl or IgG4 variable region. Once this landmark is determined, one can then locate the amino acid four residues upstream (N-terminal) to this location and replace that amino acid residue (using standard insertion/deletion methods) with a glutamic acid (E) to achieve the "Design A" modification of the disclosure. Given any variable IgGl or IgG4 immunoglobulin heavy chain amino acid query sequence of interest to use in the methods of the disclosure, one of ordinary skill in the art of antibody engineering would be able to locate the N-terminal HFR3 residue in said query sequence and then count four amino acid residues upstream therefrom to arrive at the location in HCDR2 that should be modified to glutamic acid (E).

As use herein, the phrase "a/an [amino acid name] substituted at residue in reference to a heavy chain or light chain polypeptide, refers to substitution of the parental amino acid with the indicated amino acid. For example, a heavy chain comprising "a lysine substituted at residue 39" refers to a heavy chain wherein the parental amino acid sequence has been mutated to contain a lysine at residue number 39 in place of the parental amino acid. Such mutations may also be represented by denoting a particular amino acid residue number, preceded by the parental amino acid and followed by the replacement amino acid. For example, "Q39K" refers to a replacement of a glutamine at residue 39 with a lysine. Similarly, "39K" refers to replacement of a parental amino acid with a lysine.

An antibody, Fab or other antigen binding compound of the present disclosure may be derived from a single copy or clone (e.g., a monoclonal antibody (mAb)), including any eukaryotic, prokaryotic, or phage clone. Preferably, a compound of the present disclosure exists in a homogeneous or substantially homogeneous population. In an embodiment, the antibody, Fab or other antigen binding compound, or a nucleic acid encoding the same, is provided in "isolated" form. As used herein, the term "isolated" refers to a protein, peptide or nucleic acid which is free or substantially free from other macromolecular species found in a cellular environment.

A compound of the present disclosure can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art. In particular, the methods and procedures of the Examples herein may be readily employed. An antibody, Fab or antigen binding compound of the present disclosure may be further engineered to comprise framework regions derived from fully human frameworks. A variety of different human framework sequences may be used in carrying out embodiments of the present disclosure. Preferably, the framework regions of a compound of the present disclosure are of human origin or are substantially human (at least 95%, 97% or 99% of human origin.) The sequences of framework regions of human origin may be obtained from The Immunoglobulin Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351.

Expression vectors capable of directing expression of genes to which they are operably linked are well known in the art. Expression vectors can encode a signal peptide that facilitates secretion of the desired polypeptide product(s) from a host cell. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Desired polypeptides, for example the components of the bi-specific antibodies or Fabs prepared according to the methods of the present disclosure, may be expressed independently using different promoters to which they are operably linked in a single vector or, alternatively, the desired products may be expressed independently using different promoters to which they are operably linked in separate vectors. As used herein, a "host cell" refers to a cell that is stably or transiently transfected, transformed, transduced or infected with nucleotide sequences encoding a desired polypeptide product or products. Creation and isolation of host cell lines producing a bi- specific antibody, Fab or other antigen binding compound of the present disclosure can be accomplished using standard techniques known in the art.

Mammalian cells are particular host cells for expression of the Fabs, bi-specific antibodies, or antigen binding compounds according to the present disclosure. Particular mammalian cells are HEK 293, NS0, DG-44, and CHO cells. Preferably, expressed polypeptides are secreted into the medium in which the host cells are cultured, from which the polypeptides can be recovered isolated. Medium, into which an expressed polypeptide has been secreted may be purified by conventional techniques. For example, the medium may be applied to and eluted from a Protein A or G column using conventional methods. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxy apatite chromatography. Recovered products may be immediately frozen, for example at -70 °C, or may be lyophilized.

In accordance with the present disclosure, methods have been identified for achieving assembly of particular Fabs by co-expressing nucleic acids encoding particular HC-LC pairs which contain designed residues in the interface of the heavy chain-light chain variable (VH/VL) domains and the heavy chain-light chain constant (CH1/CL) domains. More particularly, the methods of the present disclosure achieve improved specificity and, or stability in assembly of particular Fabs. Even more particular, the methods of the present disclosure allow the binding specificities and binding activities of the variable regions of two distinct therapeutic antibodies to be combined in a single bi-specific antibody compound.

Thus, the present disclosure provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said heavy chain CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W), wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); and said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D).

In a separate embodiment, the present disclosure provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said heavy chain CHI domain comprises an arginine substituted at residue 172 (172R) and a glycine substituted at residue 174 (174G); and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W), wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); and said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D).

In another embodiment, the present disclosure provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said heavy chain CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said light chain constant domain comprises a phenylalanine substituted at residue 135 (135F) and a tryptophan substituted at residue 176 (176W), wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains.

In another embodiment, the present disclosure provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CHI domain comprises a WT sequence; and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain comprises an arginine substituted at residue 38 (38R) and said light chain constant domain comprises a WT sequence, wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising the following: said first nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) and said second nucleic acid encodes a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In a separate embodiment, the present disclosure provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CHI domain comprises an aspartic acid substituted at residue 228 (228D); and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain comprises an arginine substituted at residue 38 (38R) and said light chain constant domain comprises a lysine substituted at residue 122 (122K), wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising the following: said first nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) and said second nucleic acid encodes a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

The present disclosure also provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said heavy chain CHI domain comprises a WT sequence; and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said light chain constant domain comprises a WT sequence, wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains.

More particularly, the present disclosure provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said IgG heavy chain constant CHI domain comprises an aspartic acid substituted at residue 228 (228D); and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D) and said light chain constant domain comprises a lysine substituted at residue 122 (122K), wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains.

The present disclosure also provides a method for producing a fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a heavy chain variable domain and an IgG heavy chain constant CHI domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); and (b) a second nucleic acid encoding both a light chain variable domain and a light chain constant domain wherein said light chain variable domain comprises an arginine substituted at residue 38 (38R) and said light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W), wherein each of said heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to the same antigen; (2) cultivating said host cell under conditions such that said heavy chain variable and constant domains and said light chain variable and constant domains are produced; and (3) recovering from said host cell a Fab comprising said heavy chain variable and constant domains and said light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising the following: said first nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) and said second nucleic acid encodes a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In a more particular embodiment, the present disclosure provides a method for producing a first and second fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a first heavy chain variable domain and a first IgG heavy chain constant CHI domain, wherein said first heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said first IgG heavy chain constant CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding both a first light chain variable domain and a first light chain constant domain, wherein said first light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said first light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding both a second heavy chain variable domain and a second IgG heavy chain constant CHI domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CHI domain comprises a WT sequence; and (d) a fourth nucleic acid encoding both a second light chain variable domain and a second light chain constant domain, wherein said second light chain variable domain comprises an arginine substituted at residue 38 (38R) and said second light chain constant domain comprises a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chain variable and IgG CHI constant domains and said first and second light chain variable and constant domains are produced; and (3) recovering from said host cell a first and second Fab wherein said first Fab comprises said first heavy chain variable and constant domains and said first light chain variable and constant domains, and said second Fab comprises said second heavy chain variable and constant domains and said second light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising an leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In a further embodiment, the present disclosure provides a method for producing a first and second fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a first heavy chain variable domain and a first IgG heavy chain constant CHI domain, wherein said first heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said first IgG heavy chain constant CHI domain comprises an arginine substituted at residue 172 (172R) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding both a first light chain variable domain and a first light chain constant domain, wherein said first light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said first light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding both a second heavy chain variable domain and a second IgG heavy chain constant CHI domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CHI domain comprises a WT sequence; and (d) a fourth nucleic acid encoding both a second light chain variable domain and a second light chain constant domain, wherein said second light chain variable domain comprises an arginine substituted at residue 38 (38R) and said second light chain constant domain comprises a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chain variable and IgG CHI constant domains and said first and second light chain variable and constant domains are produced; and (3) recovering from said host cell a first and second Fab wherein said first Fab comprises said first heavy chain variable and constant domains and said first light chain variable and constant domains, and said second Fab comprises said second heavy chain variable and constant domains and said second light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In a separate embodiment, the present disclosure provides A method for producing a first and second fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a first heavy chain variable domain and a first IgG heavy chain constant CHI domain, wherein said first heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said first IgG heavy chain constant CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding both a first light chain variable domain and a first light chain constant domain, wherein said first light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said first light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding both a second heavy chain variable domain and a second IgG heavy chain constant CHI domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CHI domain comprises an aspartic acid substituted at residue 228 (228D); and (d) a fourth nucleic acid encoding both a second light chain variable domain and a second light chain constant domain, wherein said second light chain variable domain comprises an arginine substituted at residue 38 (38R) and said second light chain constant domain comprises a lysine substituted at residue 122 (122K), wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chain variable and IgG CHI constant domains and said first and second light chain variable and constant domains are produced; and (3) recovering from said host cell a first and second Fab wherein said first Fab comprises said first heavy chain variable and constant domains and said first light chain variable and constant domains, and said second Fab comprises said second heavy chain variable and constant domains and said second light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In another embodiment, the present disclosure provides a method for producing a first and second fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a first heavy chain variable domain and a first IgG heavy chain constant CHI domain, wherein said first heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said first IgG heavy chain constant CHI domain comprises an arginine substituted at residue 172 (172R) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding both a first light chain variable domain and a first light chain constant domain, wherein said first light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said first light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding both a second heavy chain variable domain and a second IgG heavy chain constant CHI domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CHI domain comprises an aspartic acid substituted at residue 228 (228D); and (d) a fourth nucleic acid encoding both a second light chain variable domain and a second light chain constant domain, wherein said second light chain variable domain comprises an arginine substituted at residue 38 (38R) and said second light chain constant domain comprises a lysine substituted at residue 122 (122K), wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chain variable and IgG CHI constant domains and said first and second light chain variable and constant domains are produced; and (3) recovering from said host cell a first and second Fab wherein said first Fab comprises said first heavy chain variable and constant domains and said first light chain variable and constant domains, and said second Fab comprises said second heavy chain variable and constant domains and said second light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In another particular embodiment, the present disclosure provides a method for producing a first and second fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a first heavy chain variable domain and a first IgG heavy chain constant CHI domain, wherein said first heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and said first IgG heavy chain constant CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding both a first light chain variable domain and a first light chain constant domain, wherein said first light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and said first light chain constant domain comprises a phenylalanine substituted at residue 135 (135F) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding both a second heavy chain variable domain and a second IgG heavy chain constant CHI domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CHI domain comprises a WT sequence; and (d) a fourth nucleic acid encoding both a second light chain variable domain and a second light chain constant domain, wherein said second light chain variable domain comprises an arginine substituted at residue 38 (38R) and said second light chain constant domain comprises a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chain variable and IgG CHI constant domains and said first and second light chain variable and constant domains are produced; and (3) recovering from said host cell a first and second Fab wherein said first Fab comprises said first heavy chain variable and constant domains and said first light chain variable and constant domains, and said second Fab comprises said second heavy chain variable and constant domains and said second light chain variable and constant domains.

The present disclosure also provides a method for producing a first and second fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a first heavy chain variable domain and a first IgG heavy chain constant CHI domain, wherein said first heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y), and said first IgG heavy chain constant CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding both a first light chain variable domain and a first light chain constant domain, wherein said first light chain variable domain comprises an arginine substituted at residue 38 (38R), and said first light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding both a second heavy chain variable domain and a second IgG heavy chain constant CHI domain, wherein said second heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and said second IgG heavy chain constant CHI domain comprises a WT sequence; and (d) a fourth nucleic acid encoding both a second light chain variable domain and a second light chain constant domain, wherein said light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D) and said second light chain constant domain comprises a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chain variable and IgG CHI constant domains and said first and second light chain variable and constant domains are produced; and (3) recovering from said host cell a first and second Fab wherein said first Fab comprises said first heavy chain variable and constant domains and said first light chain variable and constant domains, and said second Fab comprises said second heavy chain variable and constant domains and said second light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising the following: said first nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) and said second nucleic acid encodes a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In another embodiment, the present disclosure provides a method for producing a first and second fragment, antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding both a first heavy chain variable domain and a first IgG heavy chain constant CHI domain, wherein said first heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y), and said first IgG heavy chain constant CHI domain comprises an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding both a first light chain variable domain and a first light chain constant domain, wherein said first light chain variable domain comprises an arginine substituted at residue 38 (38R), and said first light chain constant domain comprises a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding both a second heavy chain variable domain and a second IgG heavy chain constant CHI domain, wherein said second heavy chain variable domain comprises a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and said second IgG heavy chain constant CHI domain comprises an aspartic acid substituted at residue 228 (228D); and (d) a fourth nucleic acid encoding both a second light chain variable domain and a second light chain constant domain, wherein said light chain variable domain is a kappa isotype and comprises an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D) and said second light chain constant domain comprises a lysine substituted at residue 122 (122K), wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chain variable and IgG CHI constant domains and said first and second light chain variable and constant domains are produced; and (3) recovering from said host cell a first and second Fab wherein said first Fab comprises said first heavy chain variable and constant domains and said first light chain variable and constant domains, and said second Fab comprises said second heavy chain variable and constant domains and said second light chain variable and constant domains. More particular to this embodiment, the present disclosure provides a method comprising the following: said first nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) and said second nucleic acid encodes a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

The present disclosure also provides a method for producing a bispecific antibody comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and a CHI constant domain comprising an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding a first light chain, wherein said first light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding a second IgG heavy chain, wherein said second heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), and a CHI constant domain comprising a WT sequence; and (d) a fourth nucleic acid encoding a second light chain, wherein said second light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R) and a constant domain comprising a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second IgG heavy chains and said first and second light chains are produced; and (3) recovering from said host cell a bispecific antibody comprising a first and second fragment, antigen binding (Fab) wherein said first Fab comprises said first IgG heavy chain and said first light chain and said second Fab comprises said second IgG heavy chain and said second light chain. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In a separate embodiment, the present disclosure provides a method for producing a bispecific antibody comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is 4 amino acids upstream of the first residue of HFR3 according to Kabat, and a CHI constant domain comprising an arginine substituted at residue 172 (172R) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding a first light chain, wherein said first light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding a second IgG heavy chain, wherein said second heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), and a CHI constant domain comprising a WT sequence; and (d) a fourth nucleic acid encoding a second light chain, wherein said second light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R) and a constant domain comprising a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second IgG heavy chains and said first and second light chains are produced; and (3) recovering from said host cell a bispecific antibody comprising a first and second fragment, antigen binding (Fab) wherein said first Fab comprises said first IgG heavy chain and said first light chain and said second Fab comprises said second IgG heavy chain and said second light chain. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In yet another embodiment, the present disclosure provides a method for producing a bispecific antibody comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and a CHI constant domain comprising an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding a first light chain, wherein said first light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding a second IgG heavy chain, wherein said second heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), and a CHI constant domain comprising an aspartic acid substituted at residue 228 (228D); and (d) a fourth nucleic acid encoding a second light chain, wherein said second light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R) and a constant domain comprising a lysine substituted at residue 122 (122K), wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second IgG heavy chains and said first and second light chains are produced; and (3) recovering from said host cell a bispecific antibody comprising a first and second fragment, antigen binding (Fab) wherein said first Fab comprises said first IgG heavy chain and said first light chain and said second Fab comprises said second IgG heavy chain and said second light chain. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

In another embodiment, the present disclosure provides a method for producing a bispecific antibody comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, and a CHI constant domain comprising an arginine substituted at residue 172 (172R) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding a first light chain, wherein said first light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding a second IgG heavy chain, wherein said second heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), and a CHI constant domain comprising an aspartic acid substituted at residue 228 (228D); and (d) a fourth nucleic acid encoding a second light chain, wherein said second light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R) and a constant domain comprising a lysine substituted at residue 122 (122K), wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second IgG heavy chains and said first and second light chains are produced; and (3) recovering from said host cell a bispecific antibody comprising a first and second fragment, antigen binding (Fab) wherein said first Fab comprises said first IgG heavy chain and said first light chain and said second Fab comprises said second IgG heavy chain and said second light chain. More particular to this embodiment, the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CHI constant domain further comprising a methionine or isoleucine substituted at residue 190 (190M or 1901); said second nucleic acid encodes a light chain constant domain further comprising a leucine substituted at residue 133 (133L); said second nucleic acid encodes a light chain constant domain further comprising a glutamine or aspartic acid substituted at residue 174 (174Q or 174D), and said third nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) with said fourth nucleic acid encoding a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

The present disclosure also provides a method for producing a bispecific antibody comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), and a CHI constant domain comprising an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding a first light chain, wherein said first light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding a second IgG heavy chain, wherein said heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and a constant domain comprising a WT sequence; and (d) a fourth nucleic acid encoding second light chain, wherein said second light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second IgG heavy chains and said first and second light chains are produced; and (3) recovering from said host cell a bispecific antibody comprising a first and second fragment, antigen binding (Fab) wherein said first Fab comprises said first IgG heavy chain and said first light chain and said second Fab comprises said second IgG heavy chain and said second light chain. More particular to this embodiment, the present disclosure provides a method comprising the following: said first nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) and said second nucleic acid encodes a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

The present disclosure also provides a method for producing a bispecific antibody comprising: (1) co-expressing in a host cell: (a) a first nucleic acid encoding a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), and a CHI constant domain comprising an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G); (b) a second nucleic acid encoding a first light chain, wherein said first light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding a second IgG heavy chain, wherein said heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and a constant domain comprising an aspartic acid substituted at residue 228 (228D); and (d) a fourth nucleic acid encoding second light chain, wherein said second light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a lysine substituted at residue 122 (122K), wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second IgG heavy chains and said first and second light chains are produced; and (3) recovering from said host cell a bispecific antibody comprising a first and second fragment, antigen binding (Fab) wherein said first Fab comprises said first IgG heavy chain and said first light chain and said second Fab comprises said second IgG heavy chain and said second light chain. More particular to this embodiment, the present disclosure provides a method comprising the following: said first nucleic acid encodes a heavy chain variable domain further comprising an arginine substituted at residue 105 (105R) and said second nucleic acid encodes a light chain variable domain further comprising an aspartic acid substituted at residue 42 (42D).

As a further particular embodiment to the methods for producing a bispecific antibody, as provided herein, the present disclosure provides a method wherein one of said first and second IgG heavy chains further comprises a CH3 constant domain comprising a lysine substituted at residue 356 and a lysine substituted at residue 399, and the other of said first and second IgG heavy chains further comprises a CH3 constant domain comprising an aspartic acid substituted at residue 392 and an aspartic acid substituted at residue 409.

Even more particularly, the present disclosure provides a method for producing a bispecific antibody comprising: (1) co-expressing in ahost cell: (a) a first nucleic acid encoding a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, a CHI constant domain comprising an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G) and a CH3 constant domain comprising a lysine substituted at residue 356 (356K) and a lysine substituted at residue 399 (399K); (b) a second nucleic acid encoding a first light chain, wherein said first light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a third nucleic acid encoding a second IgG heavy chain, wherein said second heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), a CHI constant domain comprising a WT sequence and a CH3 constant domain comprising an aspartic acid substituted at residue 392 (392D) and an aspartic acid substituted at residue 409 (409D); and (d) a fourth nucleic acid encoding a second light chain, wherein said second light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R) and a constant domain comprising a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second IgG heavy chains and said first and second light chains are produced; and (3) recovering from said host cell a bispecific antibody comprising a first and second fragment, antigen binding (Fab) wherein said first Fab comprises said first IgG heavy chain and said first light chain and said second Fab comprises said second IgG heavy chain and said second light chain.

In other particular embodiments, the host cell for use in the methods of the present disclosure is a mammalian cell, more particularly a HEK293 or CHO cell, and the IgG heavy chain constant domain produced by the methods of the present disclosure is IgGl or IgG4 isotype, and more particularly IgGl .

The present disclosure also provides Fabs, bi-specific antibodies and bi-specific antigen binding compounds, each produced according to the methods of the present disclosure, as well as host cells comprising nucleic acids encoding the same. In particular, the present disclosure provides any of the Fabs, bispecific antibodies, nucleic acids or host cells as exemplified herein.

In another particular embodiment, the present disclosure provides a bispecific antibody comprising a first IgG heavy chain, wherein said first heavy chain comprises a variable domain comprising a lysine substituted at residue 39 (39K) and a glutamic acid substituted at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat, a CHI constant domain comprising an alanine substituted at residue 172 (172A) and a glycine substituted at residue 174 (174G) and a CH3 constant domain comprising a lysine substituted at residue 356 (356K) and a lysine substituted at residue 399 (399K); (b) a first light chain, wherein said first light chain comprises a kappa variable domain comprising an arginine substituted at residue 1 (1R) and an aspartic acid substituted at residue 38 (38D), and a constant domain comprising a tyrosine substituted at residue 135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) a second IgG heavy chain, wherein said second heavy chain comprises a variable domain comprising a tyrosine substituted at residue 39 (39Y), a CHI constant domain comprising a WT sequence and a CH3 constant domain comprising an aspartic acid substituted at residue 392 (392D) and an aspartic acid substituted at residue 409 (409D); and (d) a second light chain, wherein said second light chain comprises a variable domain comprising an arginine substituted at residue 38 (38R) and a constant domain comprising a WT sequence, wherein each of said first heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a first antigen and further wherein each of said second heavy chain and light chain variable domains comprise three complementarity determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen.

See also WO2016118742 and U.S. Patent Publication No. 2016039947, the entire cotents of which are hereby incorporated by reference.

F. Purification

In certain embodiments, the antibodies of the present disclosure may be purified. The term "purified," as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-poly peptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

Commonly, complete antibodies are fractionated utilizing agents (i.e. , protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies is bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. IV. Passive Immunization and Treatment/Prevention of Diabetes

A. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprising bispecific antibodies binding to CD4 and CD8. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be of any source, but in particular as high-titer humanized monoclonal antibodies (MAb). Such immunity generally lasts for a certain period of time, and further administration may be rqeuired. There is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e. , sterile and syringeable.

Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions of the disclosure can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

B. Combination Therapy

One general approach to treat disease is to combine multiple therapies as a way of increasing their efficacy. In the context of the present disclosure, the inventors propose that the bispecific antibody peptide therapy can be used successfully in conjunction with another diabetes therapeutic or regimen to treat the disease.

Using the methods and compositions of the present disclosure, one would generally contact a subject with the bispecific antibody of the present application, and another diabetes therapy. These therapies would be provided in a combined amount effective to address one or more symptom or underlying cause of diabetes. This process may involve administering both agents/therapies at the same time. This may be achieved by administering a single composition or pharmacological formulation that includes both therapies, or by using two distinct compositions or formulations, at the same time, wherein one composition includes the MUC1 peptide and the anti-ERa therapy.

Alternatively, one treatment may precede or follow the other therapy by intervals ranging from minutes to weeks. In embodiments where the therapies are applied separately, one would generally ensure that a significant period of time did not expire between each delivery, such that the therapies would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 12 hours. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. It also is conceivable that more than one administration of either the bispecific antibody or the other therapy will be desired. Various combinations may be employed, where the bispecific antibody is "A" and the other therapy is "B," as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve a therapeutic goal, both therapies are delivered to a subject in a combined amount effective to achieve that goal.

V. Kits

In still further embodiments, the present disclosure concerns kits for use with the methods described above. The kits will thus comprise, in suitable container means, a bispecific antibody that binds to CD4 and CD8, and optionally other reagents. The components of the kits may be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

VI. Examples

The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1 - Characterization and efficacy of anti-CD4 and -CD8 antibodies in animal models of Type I Diabetes and T cell-mediated pathology

ASSESSING THERAPEUTIC EFFICACY OF CORECEPTOR THERAPY IN NOD MICE. To define the long-term therapeutic effects of anti-CD4 (YTS177) and anti- CD8a (YTS105) therapy, recent onset diabetic NOD mice with blood glucose levels greater than 250 mg/dl received 2 intraperitoneal (i.p.) injections of 600-800 μg over 72 hours of each antibody within 10 days of diagnosis. YTS 177/YTS105 induces long-term remission in NOD mice determined by weekly testing of blood glucose levels, measured with a glucometer (FIG.1A). Within 72 hours of YTS177/YTS105 treatment blood glucose levels are significantly reduced (FIG. IB). Cellular infiltration of the islets (insulitis) is decreased in long-term remission (greater than 200 days) NOD mice as determined by hematoxylin and eosin staining of paraffin sections of pancreases analyzed by light microscopy (FIG 1C).

Induction of remission requires both YTS 177 and YTS105 to induce efficient remission. Treatment of newly diabetic NOD mice with 800 μg of YTS 105 (FIG. 2A) or YTS 177 (FIG. 2B) alone has no or only a limited effect on blood glucose levels and diabetes reversal.

DEFINING THE MECHANISMS OF PROTECTION OF CORECEPTOR

THERAPY IN NOD MOUSE MODELS. Induction of diabetes reversal is due to selective T cell purging (FIG. 3A). CD4+ and CD8+ T cell numbers, measured by flow cytometry, are reduced in the pancreas and draining pancreatic lymph nodes (PLN) but not the spleen of NOD mice treated with 800 μg of YTS177/YTS105 versus isotype control 2A3 antibody (FIG. 3 A). In addition to conventional T cells, immunoregulatory Foxp3+Treg are also reduced in the pancreas and PLN but not the spleen of YTS177/YTS 105-treated NOD mice (FIG. 3B).

T cell purging by coreceptor therapy is preceded by dampening of the proinflammatory milieu of the islets. NOD mice were treated with YTS 177/YTS 105 or control 2A3 antibody and over a 40 hour period, pancreatic islets isolated on a Ficoll gradient, and mRNA levels measured via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Downregulation of mRNA encoding proinflammatory cytokines and chemokines is detected as early as 6 hours post- YTS 177/YTS 105 treatment (FIG. 4B). In contrast, significant T cell purging is detected at 18 hours post-treatment. YTS 177/YTS 105-induced T cell purging is due to enhanced reactivity to sphingosine-1 -phosphate (SIP), a major regulator of T cell tissue egress. NOD mice transgenic for the BDC2.5 clonotypic T cell receptor (NOD.BDC) were treated with YTS177 or control 2A3, and 12 hours later, CD4+ T cells harvested from the PLN and spleen. Purified T cells were cultured in a transwell plate and chemotaxis in response to varying concentrations of S IP measured by flow cytometry. CD4+ T cells from the PLN but not the spleen of YTS177 treated NOD.BDC mice exhibit enhanced chemotaxis to SIP (FIG. 5B).

To determine a role for immunoregulation in YTS177/YTS105-induced protection, long-term remission NOD mice, greater than 100 days post-treatment, were injected i.p. with carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4+ T cells harvested from the spleen of NOD.BDC mice. Proliferation of BDC CD4+ T cells is reduced in the PLN of long-term remission but not control NOD recipients as determined by flow cytometry (FIG. 6B). In addition, the frequency of immunoregulatory Foxp3+Treg is increased in the PLN but not the spleen of long-term remission NOD mice as determined by flow cytometry (FIG. 7). These results are consistent with long-term protection being mediated by immunoregulation. To determine the selective effects of YTS177/YTS105 on autoreactive T cells versus T cells specific for a viral pathogen, reactivity to a lymphocytic choriomeningitis virus (LCMV) challenge was tested (FIG. 8 A). NOD mice were inoculated i.p. with 1,000 viral particles of LCMV (Armstrong) and then treated 72 hours later with 500 μg of YTS177/YTS105 or control 2A3. Seven days later, an equivalent frequency (FIG. 8B) and number (FIG. 8C) of LCMV - specific CD8+ T cells, measured by flow cytometric detection of H2Db tetramers complexed with the LCMV gp33-41 peptide, was seen. Therefore, coreceptor therapy has a minimal effect on T cell reactivity to a viral pathogen.

CHARACTERIZATION OF NONDEPLETING ANTI-HUMAN (hu)CD4 AND - huCD8a IgG4. To test the in vivo effects of engineered nondepleting anti-huCD4 and anti- huCD8a IgG4, the NRG-huPBL humanized mouse model is being used. Immunodeficient NRG mice are reconstituted with human peripheral blood mononuclear cells (PBMC) purified on a on Ficoll-hypaque gradient (FIG. 9A). Typically, within 6-8 weeks post-PBMC transfer, NRG-huPBL mice succumb to xenogeneic graft versus host disease (xGVHD) due to huCD4+ and huCD8+ T cell-driven systemic tissue destruction. Treatment of NRG-huPBL mice after huT cell reconstitution with nondepleting anti-huCD4 and anti-huCD8a IgG4 delays xGVHD monitored in part via a weight loss (FIG. 9B). In addition, coreceptor therapy reduces serum levels of human proinflammatory cytokines such as IFNy (FIG. 9C) measured by ELISA, and induces CD4+ and CD 8+ T cell purging of the pancreas and liver determined by flow cytometry (FIG. 10). T cell purging is tissue specific since splenic T cell numbers are increased following coreceptor therapy (FIG. 10). This tissue-specificity is due to the preferential effects of coreceptor therapy on tissue resident CD69+ huCD4+ and huCD8+ T cells (FIG.11). The frequency of CD69+ huT cells, indicative of recent antigen stimulation, is increased in target tissues such as the pancreas and liver relative to the spleen (FIG. 11).

CHARACTERIZATION OF NONDEPLETING BISPECIFIC ANTI-CD4 AND - CD8a ANTIBODY. Characterization of the properties of the engineered nondepleting bispecific antibodies is largely carried out as described above for the monospecific antibodies. In FIG. 12A, flow cytometric binding data is provided for the bispecific HC8.4 which has been engineered with the YTS177 and YTS105 heavy and light chain variable regions. Binding of HC8.4 to murine CD4+ and CD8+ T cells is detected via flow cytometry using an anti-rat IgG2a antibody. In addition, enhanced T cell purging of the PLN is observed in NOD mice 72 hours after treatment with HC8.4 versus YTS177/YTS105 or control 2A3 (FIG. 12B).

In vitro binding of nondepleting anti-huCD4/huCD8a bispecific antibody to huCD4+ and huCD8+ T cells is detected via flow cytometry (FIG. 13). As expected, binding of the corresponding monospecific anti-huCD4 and anti-huCD8a IgG4 to huCD4+ and huCD8+ T cells, respectively, is also detected (FIG 14). In addition, the nondepleting anti- huCD4/huCD8a bispecific antibody exhibits enhanced tissue-specific purging properties in vivo. huT cell purging is increased in the pancreas and liver of NRG-huPBL mice within 72 hours of receiving anti-huCD4/huCD8a bispecific antibody versus the combination of the monospecific anti-huCD4 and anti-huCD8a IgG4 (FIG. 17). huT cell purging by the anti- huCD4/huCD8a bispecific antibody is also tissue-specific; in contrast to the pancreas and liver splenic T cell numbers are increased (FIG. 17).

The nucleotide and amino acid sequences for the heavy and light chain variable regions used to engineer the anti-huCD4/huCD8a bispecific antibody have been determined (FIGS. 15-16).

Example 2 - CD4 Epitope Mapping (Methods)

SYNTHESIS OF PEPTIDES. To reconstruct epitopes of the target molecule a library of peptide based epitope mimics was synthesized using solid-phase Fmoc synthesis. An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with tbutyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc- peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (Perkin Elmer).

Synthesis of structural mimics was done using Pepscan's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows to structure peptides into single loops, doubleloops, triple loops, sheet-like folds, helix-like folds and combinations thereof. CLIPS templates are coupled to cysteine residues. The side-chains of multiple cysteines in the peptides are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of the P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (l :3(v/v)). This solution is added onto the peptide arrays. The CLIPS template will bind to side-chains of two cysteines as present in the solid- phase bound peptides of the peptide-arrays (455 wells plate with 3 μΐ wells). The peptide arrays are gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrays are washed extensively with excess of H20 and sonicated in disrupt- buffer containing 1% SDS/0.1 % beta-mercaptoethanol in PBS (pH 7.2) at 70°C for 30 minutes, followed by sonication in H2O for another 45 minutes. The T3 CLIPS carrying peptides were made in a similar way but now with three cysteines.

ELISA SCREENING. The binding of antibody to each of the synthesized peptides was tested in a pepscan-based ELISA. The peptide arrays were incubated with primary antibody solution (ovemight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (SBA; Table 1) for one hour at 25°C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μΐ/ml of 3 percent H2O2 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD) - camera and an image processing system.

Table 4. Details of the antibodies

Name Supplier Catalog No.

Goat anti -human HRP conjugate Southern Biotech 2010-05 DATA PROCESSING. The values obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. The results are quantified and stored into the Peplab database. Occasionally a well contains an air-bubble resulting in a false- positive value, the cards are manually inspected and any values caused by an air-bubble are scored as 0.

SYNTHESIS QUALITY CONTROL. To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel. These were screened with commercial antibodies 3C9 and 57.9 (Posthumus et al. (1990) J. Virol. 64:3304-3309)

LITERATURE REFERENCES. Timmerman et al. (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology. Langedijk et al. (201 1) J. Mol. Recognit. 20:283-299. Helical peptide arrays for lead identification and interaction site mapping, Analytical Biochemistry 417: 149-155.

BOXPLOT ANALYSIS. The box-and-whisker plot is an exploratory tool allowing displaying batches of data (Tukey 1970, 1977). Box plots give a first impression on data distribution of a given data set. Each box plot displays data set extremes (max & min), upper and lower quartiles, median and outliers (Figure 5). The main application of this boxplot is in finding the baseline levels and estimating signal over noise to allow data quality assessment.

LINEAR INTENSITY PROFILES. Throughout the report linear intensity profiles (e.g. , top panel in FIG. 26) depict a graphical representation of the intensity profile recorded for a given sample. Often overlays of intensity profiles recorded on different peptide sets are plotted in one graph to allow direct comparison between responses of a given antibody to epitope mimics of different types.

HEAT MAP ANALYSIS. A heat map is a graphical representation of data where the values taken by a variable in a two-dimensional map are represented as colors. Heatmaps can be used to represent or compare ELISA intensities recorded for sample(s) on set(s) of peptides. The magnitude of a response is color-coded and the key is included in the plot. FIG. 26 illustrates a response from a polyclonal sample recorded on a library of overlapping peptides. The response is displayed in two complementary ways: as histogram (top panel FIG. 26) and a heatmap (bottom panel FIG. 26). While for a single sample the histogram is easily grasped, a comparison of responses from numerous samples is easier, when analyzed in a heatmap, where each sample is plotted as a row (or column) and each peptide as a column (or row).

DESIGN OF PEPTIDES. Different sets of peptides were synthesized (see below) according to the following designs. Note that actual order of peptides on mini-cards in some was randomized. Note that not all experimental datasets yielded optimal results. These sets are not mentioned in detail in the results section, but are provided in the raw data file.

SET 1

Mimic Type Linear

Label LIN

Description Linear peptides of length 15 derived from the target sequence of

CD4 with an offset of one residue.

Sequences GPTSPKLMLSLKLEN (SEQ ID NO: 27)

(first 10) PTSPKLMLSLKLENK (SEQ ID NO: 28)

TSPKLMLSLKLENKE (SEQ ID NO: 29)

SPKLMLSLKLENKEA (SEQ ID NO: 30)

PKLMLSLKLENKEAK (SEQ ID NO: 31)

KLMLSLKLENKEAKV (SEQ ID NO: 32)

LMLSLKLENKEAKVS (SEQ ID NO: 33)

MLSLKLENKEAKVSK (SEQ ID NO: 34)

LSLKLENKEAKVSKR (SEQ ID NO: 35)

SLKLENKEAKVSKRE (SEQ ID NO: 36)

Mimic Type Linear

Label LIN.2

Description Peptides of set 1, but with Cys residues replaced by Cys

acetamydomethyl (acm; denoted "2").

Sequences KVVLGKKGDTVELT2 (SEQ ID NO: 37)

(first 10) VLGKKGDTVELT2TA (SEQ ID NO: 38)

GKKGDTVELT2TASQ (SEQ ID NO: 39)

KGDTVELT2TASQKK (SEQ ID NO: 40)

DTVELT2TASQKKSI (SEQ ID NO: 41)

VELT2TAS QKKSIQF (SEQ ID NO: 42)

LT2TASQKKSIQFHW (SEQ ID NO: 43)

2TASQKKSIQFHWKN (SEQ ID NO: 44)

IKNLKIEDSDTYI2E (SEQ ID NO: 45)

NLKIEDSDTYI2EVE (SEQ ID NO: 46) SET 3

Mimic Type Linear

Label LIN.AA

Description Peptides of set 1, but with residues on positions 10 and 11 replaced by Ala. When a native Ala would occur on either position, it is replaced by Gly.

Sequences FSFPLAFTVAALTGS (SEQ ID NO: 47)

(first 10) FPLIIKNLKAADSDT (SEQ ID NO: 48)

QGNFPLIIKAAKIED (SEQ ID NO: 49)

EVSVKRVTQAAKLQM (SEQ ID NO: 50)

LMLSLKLENAAAKVS (SEQ ID NO: 51)

TGKLHQEVNAAVMRA (SEQ ID NO: 52)

SKSWITFDLAAKEVS (SEQ ID NO: 53)

KREKAVWVLAAEAGM (SEQ ID NO: 54)

SVQCRSPRGAAIQGG (SEQ ID NO: 55)

ALPQYAGSGAATLAL (SEQ ID NO: 56)

SET 4

Mimic Type Monoloop, mP2 CLIPS

Label LOOP

Description Looped peptides of length 17. On positions 2 - 16 are 15-mer peptides derived from the target sequence of CD4 with an offset of two residues. Cys are inserted on positions 1 and 17 and joined by means of mP2 CLIPS to create a looped structure. Native Cys are replaced by Cys- acm (denoted "2").

Sequences CKKVVLGKKGDTVELTC (SEQ ID NO: 57)

(first 10) CVVLGKKGDTVELT2TC (SEQ ID NO: 58)

CLGKKGDTVELT2TASC (SEQ ID NO: 59)

CKKGDTVELT2TASQKC (SEQ ID NO: 60)

CGDTVELT2TASQKKSC (SEQ ID NO: 61)

CTVELT2TASQKKSIQC (SEQ ID NO: 62)

CELT2TASQKKSIQFHC (SEQ ID NO: 63)

CT2TAS QKKSIQFHWKC (SEQ ID NO: 64)

CTASQKKSIQFHWKNSC (SEQ ID NO: 65) CSQKKSIQFHWKNSNQC (SEQ ID NO: 66)

SET 5

Mimic Type a-helix, mP2 CLIPS

Label HEL

Description a-helical epitope mimics of length of length 22 derived from residues 72-100 of the target sequence with an offset of one residue. Cys are inserted on positions 1 and 5 and joined by means of mP2 CLIPS to nucleate an α-helical structure. Native Cys are replaced by Cys- acm (denoted "2").

Sequences CSLWCQGNFPLIIKNLKIEDSD (SEQ ID NO: 67)

(first 10) CGNFCLIIKNLKIEDSDTYI2E (SEQ ID NO: 68)

CPSKCNDRADSRRSLWDQGNFP (SEQ ID NO: 69) CQGNCPLIIKNLKIEDSDTYI2 (SEQ ID NO: 70)

CKLNCRADSRRSLWDQGNFPLI (SEQ ID NO: 71)

CGPSCLNDRADSRRSLWDQGNF (SEQ ID NO: 72) CWDQCNFPLIIKNLKIED SDTY (SEQ ID NO: 73)

CLNDCADSRRSLWDQGNFPLII (SEQ ID NO: 74)

CADSCRSLWDQGNFPLIIKNLK (SEQ ID NO: 75)

CDQGCFPLIIKNLKIEDSDTYI (SEQ ID NO: 76)

SET 6

Mimic Type β-turn, mP2 CLIPS

Label BET

Description β-turn epitope mimics of length of length 22. On positions 2 - 21 are 20-mer peptides derived from the target sequence of CD4 with an offset of one residue. Residues on positions 11 and 12 are replaced by the "PG" motif to nucleate the β-turn formation. Cys are inserted on positions 1 and 22 and joined by means of mP2 CLIPS to stabilize the β-turn structure. Native Cys are replaced by Cys- acm (denoted "2").

Sequences CKKVVLGKKGPGVELT2TASQC (SEQ ID NO: 77) (first 10) CKVVLGKKGDPGELT2TASQKC (SEQ ID NO: 78)

CVVLGKKGDTPGLT2TASQKKC (SEQ ID NO: 79) CVLGKKGDTVPGT2TASQKKSC (SEQ ID NO: 80)

CLGKKGDTVEPG2TASQKKSIC (SEQ ID NO: 81)

CGKKGDTVELPGTASQKKSIQC (SEQ ID NO: 82)

CKKGDTVELTPGASQKKSIQFC (SEQ ID NO: 83)

CKGDTVELT2P GS QKKSIQFHC (SEQ ID NO: 84)

CGDTVELT2TPGQKKSIQFHWC (SEQ ID NO: 85)

CDTVELT2TAPGKKSIQFHWKC (SEQ ID NO: 86)

SET 7

Mimic Type disulfide bridges

Label CYS.A

Description Disulfide bridge epitope mimics of length of length 21. On positions 1-9 and 13-21 are 9-mer peptides derived from the target sequence of CD4 with an offset of one residue and joined via the "GGG" linker. Only sequences containing pairing Cys (as per uniprot) are used for creating these mimics. Cys not participating in the disulfide bridge formation, but still present within a mimic, are replaced by Cys- acm (denoted "2").

Sequences KGDTVELTCGGGIEDSDTYIC (SEQ ID NO: 87)

(first 10) DTVELTCTAGGGIEDSDTYIC (SEQ ID NO: 88)

VELTCTASQGGGIEDSDTYIC (SEQ ID NO: 89)

LTCT AS QKKGGGIED SDTYIC (SEQ ID NO: 90)

CTASQKKSIGGGIEDSDTYIC (SEQ ID NO: 91)

KGDTVELTCGGGDSDTYICEV (SEQ ID NO: 92)

DTVELTCTAGGGDSDTYICEV (SEQ ID NO: 93)

VELTCTASQGGGDSDTYICEV (SEQ ID NO: 94)

LTCTASQKKGGGDSDTYICEV (SEQ ID NO: 95)

CTASQKKSIGGGDSDTYICEV (SEQ ID NO: 96) Antibody binding depends on a combination of factors, including concentration of the antibody and the amounts and nature of competing proteins in the ELISA buffer. Also, the pre-coat conditions (the specific treatment of the peptide arrays prior to incubation with the experimental sample) affect binding. These details are summed up in Table 2. For the Pepscan Buffer and Preconditioning (SQ), the numbers indicate the relative amount of competing protein (a combination of horse serum and ovalbumin).

Table 5. Screening conditions

Label Dilution Sample Buffer Pre-Conditioning

CD4 mab 1 μg/ml 25%SQ 25%SQ

CD4 mab 2 μg/ml 0.1%SQ 1%SQ

Example 3 - CD4 Epitope Mapping (Results) LINEAR EPITOPE MAPPING WITH ALL OVERLAPPING PEPTIDES. The concept of mapping linear epitopes using libraries of overlapping synthetic peptides was pioneered by Pepscan founders Geysen and Meloen (PNAS, 1984). As the inventor of the technology Pepscan has long standing expertise in addressing linear epitopes by directly synthesizing libraries of linear peptides on a solid support covered with a proprietary hydrogel formulation, which allows working with biomolecules and can be easily regenerated for profiling big sample sets. To generate a library of linear mimics, the correct amino acid sequence of the immunogen (or target protein) is split in overlapping fragments in silico, which are then synthesized on a solid support as shown in FIG. 22.

THE PRINCIPLES OF CLIPS TECHNOLOGY. The majority of biomolecules of therapeutic interest recognize conformational or discontinuous epitopes on their cognate target. To mimic this situation in peptides derived from the target, CLIPS technology structurally fixes peptides into defined 3D structures. The CLIPS reaction takes place between bromo groups of the CLIPS scaffold and thiol sidechains of cysteines introduced into peptide constructs. The reaction is ultra-fast, very specific and is undertaken under mild conditions. Using this elegant chemistry, native protein sequences are transformed into CLIPS constructs with a range of structures (FIG. 23). CLIPS technology is now routinely used to shape peptide libraries into single, double or triple looped structures as well as sheet- and helix-like folds, which allows mimicking of conformational and discontinuous binding sites.

CLIPS LIBRARY FOR PROFILING CONFORMATIONAL EPITOPES. Conformational epitopes can be mimicked using CLIPS chemistry. Simple secondary structure mimics can be stabilized by application of CLIPS scaffolds that allow selecting thermodynamically-favored peptide conformations. CLIPS peptide libraries can mimic secondary structure elements, such as loops, a-helixes and β-strands. A schematic of this approach is drawn in FIG. 24, where a single loop element is mimicked using mP2 CLIPS chemistry.

PRIMARY EXPERIMENTAL RESULTS AND SIGNAL TO NOISE RATIO DETERMINATION. The raw ELISA results of the screening are provided in the accompanying Excel data file. A graphical overview of the complete dataset is given in FIG. 27. Here a box plot depicts each dataset and indicates the average ELISA signal, the distribution and the outliers within each dataset. Depending on experiment conditions (amount of antibody, blocking strength, etc.) different distributions of ELISA data are obtained.

ANTIBODY CD4 MAB. Initially antibody CD4 was tested under high stringency conditions and did not yield any detectable binding on the array. Therefore, the antibody was re-tested under low stringency conditions and recorded binding profiles did not show any systematic binding. Data recorded either under low or high stringency conditions do not allow reliably identifying a linear or a simple conformational epitope. Examples of intensity profiles are shown in FIGS. 28-30.

CONCLUSIONS. The monoclonal antibody CD4 mAb was repeatedly tested on the array. While no binding was recorded under high stringency conditions, non-systematic binding profiles were recorded under low stringency conditions. Absence of systematic binding indicates that recorded intensity profiles most likely result from non-specific binding events. Therefore, recorded data lack the reliability sufficient for deriving an epitope candidate. It is possible that CD4 mAb binding relies structural complexity that was not addressed by the current library of epitope mimics.

Example 4 - CD8 Epitope Mapping (Methods) SYNTHESIS OF PEPTIDES. To reconstruct epitopes of the target molecule a library of peptide based epitope mimics was synthesized using solid-phase Fmoc synthesis. An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc- peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (Perkin Elmer). Synthesis of structural mimics was done using Pepscan's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows to structure peptides into single loops, double loops, triple loops, sheet-like folds, helix-like folds and combinations thereof. CLIPS templates are coupled to cysteine residues. The side-chains of multiple cysteines in the peptides are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of the P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (l :3(v/v)). This solution is added onto the peptide arrays. The CLIPS template will bind to side-chains of two cysteines as present in the solid- phase bound peptides of the peptide-arrays (455 wells plate with 3 μΐ wells). The peptide arrays are gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrays are washed extensively with excess of H20 and sonicated in disrupt- buffer containing 1% SDS/0.1 % beta-mercaptoethanol in PBS (pH 7.2) at 70°C for 30 minutes, followed by sonication in H20 for another 45 minutes. The T3 CLIPS carrying peptides were made in a similar way but now with three cysteines.

ELISA SCREENING. The binding of antibody to each of the synthesized peptides was tested in a pepscan-based ELISA. The peptide arrays were incubated with primary antibody solution (ovemight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (SBA; Table 4) for one hour at 25°C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μΐ/ml of 3 percent H202 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD) - camera and an image processing system.

Table 4. Details of the antibodies

Name Supplier Catalog No.

goat anti -human HRP conjugate Southern Biotech 2010-05

DATA PROCESSING. The values obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. The results are quantified and stored into the Peplab database. Occasionally a well contains an air-bubble resulting in a false- positive value, the cards are manually inspected and any values caused by an air-bubble are scored as 0.

SYNTHESIS QUALITY CONTROL. To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel. These were screened with commercial antibodies 3C9 and 57.9 (ref. Posthumus et al. (1990) J. Virol. 64:3304-3309)

LITERATURE REFERENCES. Timmerman et al. (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology. J. Mol. Recognit. 20:283-299 Langedijk et al. (2011). Helical peptide arrays for lead identification and interaction site mapping, Analytical Biochemistry 417: 149-155.

BOXPLOT ANALYSIS. The box-and-whisker plot is an exploratory tool allowing displaying batches of data (Tukey 1970, 1977). Box plots give a first impression on data distribution of a given data set. Each box plot displays data set extremes (max & min), upper and lower quartiles, median and outliers (FIG. 25). The main application of this boxplot is in finding the baseline levels and estimating signal over noise to allow data quality assessment.

LINEAR INTENSITY PROFILES. Throughout the report linear intensity profiles (e.g. , top panel in FIG. 32 depict a graphical representation of the intensity profile recorded for a given sample. Often overlays of intensity profiles recorded on different peptide sets are plotted in one graph to allow direct comparison between responses of a given antibody to epitope mimics of different types.

HEAT MAP ANALYSIS. A heat map is a graphical representation of data where the values taken by a variable in a two-dimensional map are represented as colors. Heatmaps can be used to represent or compare ELISA intensities recorded for sample(s) on set(s) of peptides. The magnitude of a response is color-coded and the key is included in the plot. FIG. 32 below illustrates a response from a polyclonal sample recorded on a library of overlapping peptides. The response is displayed in two complementary ways: as histogram (top panel FIG. 32) and a heatmap (bottom panel FIG. 32). While for a single sample the histogram is easily grasped, a comparison of responses from numerous samples is easier, when analyzed in a heatmap, where each sample is plotted as a row (or column) and each peptide as a column (or row).

DESIGN OF PEPTIDES. Different sets of peptides were synthesized (see also Methods section) according to the following designs. Note that actual order of peptides on mini-cards in some was randomized. Note that not all experimental datasets yielded optimal results. These sets are not mentioned in detail in the results section, but are provided in the raw data file. SET 1

Mimic Type Linear

Label LIN.8

Description Linear peptides of length 15 derived from the target sequence of

CD8 with an offset of one residue.

Sequences SQFRVSPLDRTWNLG (SEQ ID NO: 97)

(first 10) QFRVSPLDRTWNLGE (SEQ ID NO: 98)

FRVSPLDRTWNLGET (SEQ ID NO: 99)

RVSPLDRTWNLGETV (SEQ ID NO: 100)

VSPLDRTWNLGETVE (SEQ ID NO: 101)

SPLDRTWNLGETVEL (SEQ ID NO: 102)

PLDRTWNLGETVELK (SEQ ID NO: 103)

LDRTWNLGETVELKC (SEQ ID NO: 104)

DRTWNLGETVELKCQ (SEQ ID NO: 105)

RTWNLGETVELKCQV (SEQ ID NO: 106)

SET 2

Mimic Type Linear

Label LIN.28

Description Peptides of set 1, but with Cys residues replaced by Cys- acetamydomethyl (acm; denoted "2").

Sequences LDRTWNLGETVELK2 (SEQ ID NO: 107)

(first 10) RTWNLGETVELK2QV (SEQ ID NO: 108)

WNLGETVELK2QVLL (SEQ ID NO: 109)

LGETVELK2QVLLSN (SEQ ID NO: 110)

ETVELK2QVLLSNPT (SEQ ID NO: 111)

VELK2QVLLSNPTSG (SEQ ID NO: 112)

LK2QVLLSNPTSG2S (SEQ ID NO: 113)

2QVLLSNPTSG2SWL (SEQ ID NO: 114)

VLLSNPTSG2SWLFQ (SEQ ID NO: 115)

LSNPTSG2SWLFQPR (SEQ ID NO: 116)

SET 3

Mimic Type Linear Label LINAA8

Description Peptides of set 1, but with residues on positions 10 and 11 replaced by Ala. When a native Ala would occur on either position, it is replaced by Gly.

Sequences SQPLSLRPEGARPAA (SEQ ID NO: 117)

(first 10) LSLRPEACRAGAGGA (SEQ ID NO: 118)

EGLDTQRFSAARLGD (SEQ ID NO: 119)

YF SHF VP VF AAAKPT (SEQ ID NO: 120)

TTPAPRPPTAGPTIA (SEQ ID NO: 121)

HFVPVFLPAAATTTP (SEQ ID NO: 122)

KPTTTPAPRAATPAP (SEQ ID NO: 123)

PTFLLYLSQAAPKAA (SEQ ID NO: 124)

SWLFQPRGAGGSPTF (SEQ ID NO: 125)

SDFRRENEGAAFCSA (SEQ ID NO: 126)

SET 4

Mimic Type Monoloop, mP2 CLIPS

Label LOOP.8

Description Looped peptides of length 17. On positions 2 - 16 are 15-mer peptides derived from the target sequence of CD8 with an offset of one residue. Cys are inserted on positions 1 and 17 and joined by means of mP2 CLIPS to create a looped structure. Native Cys are replaced by Cys- acm (denoted "2").

Sequences CSQFRVSPLDRTWNLGC (SEQ ID NO: 127)

(first 10) CFRVSPLDRTWNLGETC (SEQ ID NO: 128)

CVSPLDRTWNLGETVEC (SEQ ID NO: 129)

CPLDRTWNLGETVELKC (SEQ ID NO: 130)

CDRTWNLGETVELK2QC (SEQ ID NO: 131)

CTWNLGETVELK2QVLC (SEQ ID NO: 132)

CNLGETVELK2QVLLSC (SEQ ID NO: 133)

CGETVELK2QVLLSNPC (SEQ ID NO: 134)

CTVELK2QVLLSNPTSC (SEQ ID NO: 135)

CELK2QVLLSNPTSG2C (SEQ ID NO: 136)

SET 5 Mimic Type a-helix, mP2 CLIPS

Label HEL.8

Description a-helical epitope mimics of length of length 22 derived from the target sequence of CD8 with an offset of one residue. Cys are inserted on positions 1 and 5 and joined by means of mP2 CLIPS to nucleate an a-helical structure. Native Cys are replaced by Cys- acm (denoted "2").

Sequences CVLTCSDFRRENEGYYF2SALS (SEQ ID NO: 137)

(first 10) CLTLCDFRRENEGYYF2SALSN (SEQ ID NO: 138)

CTLSCFRRENEGYYF2SALSNS (SEQ ID NO: 139)

CLSDCRRENEGYYF2SALSNSI (SEQ ID NO: 140)

CSDFCRENEGYYF2SALSNSIM (SEQ ID NO: 141)

CDFRCENEGYYF2SALSNSIMY (SEQ ID NO: 142) CFRRCNEGYYF2SALSNSIMYF (SEQ ID NO: 143)

CRRECEGYYF2SALSNSIMYFS (SEQ ID NO: 144)

CRENCGYYF2SALSNSIMYFSH (SEQ ID NO: 145)

CENECYYF2SALSNSIMYFSHF (SEQ ID NO: 146)

SET 6

Mimic Type β-turn, mP2 CLIPS

Label BET.8

Description β-turn epitope mimics of length of length 22. On positions 2 -

21 are 20-mer peptides derived from the target sequence of CD8 with an offset of one residue. Residues on positions 11 and 12 are replaced by the "PG" motif to nucleate the β-turn formation.

Cys are inserted on positions 1 and 22 and joined by means of mP2 CLIPS to stabilize the β-turn structure. Native Cys are replaced by Cys- acm (denoted "2").

Sequences CSQFRVSPLDPGWNLGETVELC (SEQ ID NO: 147) (first 10) CQFRVSPLDRPGNLGETVELKC (SEQ ID NO: 148)

CFRVSPLDRTPGLGETVELK2C (SEQ ID NO: 149)

CRVSPLDRTWPGGETVELK2QC (SEQ ID NO: 150) CVSPLDRTWNPGETVELK2QVC (SEQ ID NO: 151) CSPLDRTWNLPGTVELK2QVLC (SEQ ID NO: 152) CPLDRTWNLGPGVELK2QVLLC (SEQ ID NO: 153)

CLDRTWNLGEPGELK2QVLLSC (SEQ ID NO: 154) CDRTWNLGETPGLK2QVLLSNC (SEQ ID NO: 155) CRTWNLGETVPGK2QVLLSNPC (SEQ ID NO: 156)

SET 7

Mimic Type disulfide bridges

Label CYS.A8

Description Disulfide bridge epitope mimics of length of length 21. On positions 1-9 and 13-21 are 9-mer peptides derived from the target sequence of CD8 with an offset of one residue and joined via the "GGG" linker. Only sequences containing pairing Cys (as per uniprot) are used for creating these mimics. Cys not participating in the disulfide bridge formation, but still present within a mimic, are replaced by Cys- acm (denoted "2").

Sequences LGETVELKCGGGRENEGYYFC (SEQ ID NO: 157)

(first 10) ETVELKCQVGGGRENEGYYFC (SEQ ID NO: 158)

VELKCQVLLGGGRENEGYYFC (SEQ ID NO: 159) LKCQVLLSNGGGRENEGYYFC (SEQ ID NO: 160) CQVLLSNPTGGGRENEGYYFC (SEQ ID NO: 161)

LGETVELKCGGGNEGYYFCSA (SEQ ID NO: 162)

ETVELKCQVGGGNEGYYFCSA (SEQ ID NO: 163) VELKCQVLLGGGNEGYYFCSA (SEQ ID NO: 164) LKCQVLLSNGGGNEGYYFCSA (SEQ ID NO: 165) CQVLLSNPTGGGNEGYYFCSA (SEQ ID NO: 166)

SCREENING DETAILS. Antibody binding depends on a combination of factors, including concentration of the antibody and the amounts and nature of competing proteins in the ELISA buffer. Also, the pre-coat conditions (the specific treatment of the peptide arrays prior to incubation with the experimental sample) affect binding. These details are summed up in Table 5. For the Pepscan Buffer and Preconditioning (SQ), the numbers indicate the relative amount of competing protein (a combination of horse serum and ovalbumin).

Table 5. Screening conditions Label Dilution Sample Buffer Pre-conditioning

CD8 mab 1 μg/ml 25%SQ 25%SQ

CD8 mab 5 μg/ml 1$SQ 1%SQ

Example 5 - CD8 Epitope Mapping (Results)

LINEAR EPITOPE MAPPING WITH ALL OVERLAPPING PEPTIDES. The concept of mapping linear epitopes using libraries of overlapping synthetic peptides was pioneered by Pepscan founders Geysen and Meloen (PNAS, 1984). As the inventor of the technology Pepscan has long standing expertise in addressing linear epitopes by directly synthesizing libraries of linear peptides on a solid support covered with a proprietary hydrogel formulation, which allows working with biomolecules and can be easily regenerated for profiling big sample sets. To generate a library of linear mimics, the correct amino acid sequence of the immunogen (or target protein) is split in overlapping fragments in silico, which are then synthesized on a solid support as shown in FIG. 22.

THE PRINCIPLES OF CLIPS TECHNOLOGY. The majority of biomolecules of therapeutic interest recognize conformational or discontinuous epitopes on their cognate target. To mimic this situation in peptides derived from the target, CLIPS technology structurally fixes peptides into defined 3D structures. The CLIPS reaction takes place between bromo groups of the CLIPS scaffold and thiol sidechains of cysteines introduced into peptide constructs. The reaction is ultra-fast, very specific and is undertaken under mild conditions. Using this elegant chemistry, native protein sequences are transformed into CLIPS constructs with a range of structures (FIG. 23). CLIPS technology is now routinely used to shape peptide libraries into single, double or triple looped structures as well as sheet- and helix-like folds, which allows mimicking of conformational and discontinuous binding sites.

CLIPS LIBRARY FOR PROFILING CONFORMATIONAL EPITOPES.

Conformational epitopes can be mimicked using CLIPS chemistry. Simple secondary structure mimics can be stabilized by application of CLIPS scaffolds that allow selecting thermodynamically-favored peptide conformations. CLIPS peptide libraries can mimic secondary structure elements, such as loops, a-helixes and β-strands. A schematic of this approach is drawn in FIG. 24, where a single loop element is mimicked using mP2 CLIPS chemistry.

PRIMARY EXPERIMENTAL RESULTS AND SIGNAL TO NOISE RATIO DETERMINATION. The raw ELISA results of the screening are provided in the accompanying Excel data file. A graphical overview of the complete dataset is given in FIG. 33. Here a box plot depicts each dataset and indicates the average ELISA signal, the distribution and the outliers within each dataset. Depending on experiment conditions (amount of antibody, blocking strength, etc.) different distributions of ELISA data are obtained.

ANTIBODY CD8 MAB. Antibody CD8 mAb was tested on the array under high stringency conditions. Recorded intensity profiles did not display any defined peaks. Therefore, the antibody was re-tested under low stringency conditions. Detectable and systematic binding was recorded with linear, single loop and disulfide bridge epitope mimics (FIGS. 34-35). In detail, antibody CD8 mAb systematically recognized linear and disulfide bridged peptides with core sequence 109 NEGYYFCSA 117. Additionally, screening the antibody with looped peptides yielded an intensity profile with a single peak corresponding to peptides with core sequence 60 PRGAAASPTFLLY 72.

Monoclonal antibody CD8 mAb was tested on peptide libraries composed of linear and simple conformational epitope mimics. It was not possible to establish binding of the antibody under high stringency conditions, so screening the antibody under low stringency conditions was attempted. Antibody CD8 mAb yielded detectable binding on all peptide sets under low stringency conditions. Individual intensity profiles were analyzed to assess systematicity of the binding. The antibody bound peptides with core sequence 109 NEGYYFCSA 117 in three different peptide sets - linear, β-turn and disulfide bridges.

Binding to peptides with core sequence 60 PRGAAASPTFLLY 72 was only established in peptide set containing single loop peptides, lakj.pdb was used to assess structural features of identified epitopes (FIGS. 36-37). Even though 60 PRGAAASPTFLLY 72 and 109 NEGYYFCSA 117 are separated at the level of the primary structure, they form a continuous surface in 3D space. Moreover, both partial epitopes cover the dimerization interface of the CD8 molecule.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

VII. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

WHAT IS CLAIMED IS;

1. A bispecific antibody or antibody fragment having binding specificity for CD4 and CD8.

The antibody of claims 1, wherein said antibody comprises heavy chain CDRs of SEQ ID NOS: 1-3 and light chain CDRs of SEQ ID NOS: 4-6, and heavy chain CDRs of SEQ ID NOS: 7-9 and light chain CDRs of SEQ ID NOS: 10-12.

The antibody of claims 1-2, wherein the antibody or antibody fragment is encoded by heavy and light chain variable sequences as set forth in SEQ ID NOS: 14, 16, 18, and 20.

4. The antibody of claims 1-3, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 70%, 80%, or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 14, 16, 18, and 20.

5. The antibody of claims 1-3, wherein said antibody or antibody fragment is encoded by heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 14, 16, 18, and 20.

6. The antibody of claims 1-3, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences heavy and light chain variable sequences as set forth in SEQ ID NOS: 13, 15, 17, and 19.

7. The antibody of claims 1-3, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 70%, 80% or 90% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 13, 15, 17, and 19.

8. The antibody of claims 1-3, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having 95% identity to heavy and light chain variable sequences as set forth in SEQ ID NOS: 13, 15, 17, and 19.

9. The antibody of claims 1-8, wherein the antibody fragment is a recombinant antibody, F(ab')2 fragment, or Fv fragment.

10. The antibody of claims 1-9, wherein said antibody is a chimeric antibody, a human antibody, an IgG antibody or a humanized antibody.

11. A bispecific antibody or antibody fragment having binding specificity for CD4 and CD8, wherein said antibody recognizes a discontinuous epitope found in CD8 residues NEGYYFCSA and/or PRGAAASPTFLLY.

12. The antibody of claim 11, wherein the antibody fragment is a recombinant antibody, F(ab')2 fragment, or Fv fragment.

13. The antibody of claims 11-12, wherein said antibody is a chimeric antibody, a human antibody, an IgG antibody or a humanized antibody.

14. A method of treating diabetes in a subject comprising providing an antibody or antibody fragment according to claims 1-13 to said subject.

15. The method of claim 13, wherein diabetes is type 1 diabetes.

16. The method of claim 13, wherein the subject is a human.

17. The method of claim 13, wherein the subject is a non-human mammal.

18. The method of claims 13-17, wherein the providing is chronic, such as daily, weekly, monthly, every other month, every three months, every four months, every five months, every six months, every nine months or every year.

19. The method of claims 13-18, wherein the effects of providing are persistent.

20. The method of claims 13-19, wherein providing comprises administering said antibody or antibody fragment to said antibody.

21. The method of claims 13-19, wherein providing comprises genetic delivery of an RNA or DNA sequence or vector encoding the antibody or antibody fragment.

22. The method of claims 1 1-21 , wherein providing results in reducing T effector cell number in the subject's pancreas and/or pancreatic lymph nodes, such as CD4⁺ T cells.

23. The method of claims 11 -21 , wherein providing results in an increase in Fox3⁺ Treg cell number and/or activity in the subject's pancreas and/or pancreatic lymph nodes.

24. The method of claims 11 -23, further comprising administering to said subject a second diabetes therapy.

25. The method of claims 11 -20, wherein said subject is selected from a neonate, an pediatric patient, a teenager, an adult or a patient over about 60 years of age.

26. A hybridoma or engineered cell encoding an antibody or antibody fragment according to claims 1 -13.

27. A vaccine formulation comprising one or more antibodies or antibody fragments according to claims 1-13.

28. A kit comprising an antibody or antibody fragment according to claims 1-13.