WO2020060924A1 - Use of a cd4/cd8 bispecific antibody for the treatment of autoimmune/inflammatory disorders - Google Patents
Use of a cd4/cd8 bispecific antibody for the treatment of autoimmune/inflammatory disorders Download PDFInfo
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Definitions
- 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.
- An autoimmune disease is a condition arising from an abnormal immune response to a normal body part, often starting in young adulthood. There are at least 80 types of autoimmune diseases. Nearly any body part can be involved. Common symptoms include low grade fever and feeling tired. Often symptoms come and go. About 24 million (7%) people in the United States are affected by an autoimmune disease. Women are more commonly affected than men, and it has been estimated that autoimmune diseases are among the leading causes of death among women in the United States in all age groups up to 65 years.
- autoimmune diseases such as lupus run in families, and certain cases may be triggered by infections or other environmental factors.
- Some common autoimmune diseases include celiac disease, diabetes mellitus type 1, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. The diagnosis can be difficult to determine given the similar and overlapping symptoms, and the distinguishing causative factors for each of these diseases are still being sorted out.
- Nonsteroidal anti inflammatory drugs NSAIDs
- immunosuppressants are often used.
- Intravenous Immunoglobulin may also occasionally be used. While treatment usually improves symptoms, they do not typically cure the disease. A further challenge is being able to tailor a response to the particular aspects of an individual’s disease.
- NSAIDs nonsteroidal anti inflammatory drugs
- IPDs immunosuppressants
- Treatment usually improves symptoms, they do not typically cure the disease.
- a further challenge is being able to tailor a response to the particular aspects of an individual’s disease.
- a therapeutic approach that was valid for all subject’s with a given autoimmune disease would be extremely valuable, and an approach that was valid for multiple clinically distinct autoimmune diseases would be a remarkable advance in the care of patients afflicted with these maladies.
- antibodies targeted to CD4 and CD8 are provided herein.
- a bispecific antibody or antibody fragment having binding specificity for CD4 and CD8.
- 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.
- 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.
- 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.
- 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 fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’) 2 fragment, or Fv fragment.
- antibody is a chimeric antibody, a human antibody, an IgG antibody or a humanized antibody.
- 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 (SEQ ID NO: 168) and/or PRGAAASPTFLLY (SEQ ID NO: 167).
- the antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’) 2 fragment, or Fv fragment.
- 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 pattern, 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.
- a method of treating an autoimmune/inflammatory disease 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.
- the subject is a human.
- the subject is a non-human mammal.
- the subject is selected from a neonate, a pediatric patient, a teenager, an adult or a patient over about 60 years of age.
- 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.
- the effects of providing are persistent.
- providing comprises administering said antibody or antibody fragment to said antibody.
- providing comprises genetic delivery of an RNA or DNA sequence or vector encoding the antibody or antibody fragment.
- providing results in reducing T effector cell number in any of the subject’s inflamed tissues including but not limited to the liver, pancreas, salivary glands, ovaries, testes, skin, central nervous system, synovial tissue, gastrointestinal tract, thyroid, kidneys, lungs or eyes, such as CD4 + or CD8 + T cells.
- the method further comprises administering to said subject a second autoimmune/anti-inflammatory therapy.
- 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).
- 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).
- T cell refers to T lymphocytes, and includes, but is not limited to, gd + T cells, NK T cells, CD4 + T cells and CD8 + T cells.
- CD4 + T cells include THO, THI and TH2 cells, as well as regulatory T cells (Treg). There are at least three types of regulatory T cells: CD4 + CD25 + Treg, CD25 TH3 T reg , and CD25 TRT T re g.“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 preferred 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
- 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.
- 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).
- 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.
- a monoclonal antibody 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.
- 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.
- 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.
- 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.
- each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
- monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
- A“non-depleting antibody”, such as a mAh 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.
- 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.
- FIG. 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. 1B 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.
- FIG. 2A-2B Both nondepleting anti-CD4 and -CD8 antibodies are needed for efficient diabetes remission.
- FIG. 3A-3B Induction of remission correlates with T cell purging of the pancreas.
- FIG. 3 A 6 days post-YTSl77/YTSl05 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 2A3 treated NOD mice.
- FIG. 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 2 A3 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.
- FIG. 5 A Schematic of experimental approach.
- FIG. 5B Percent migration to S1P in an in vitro transwell assay of CD4+ T cells from the spleen or PLN of NOD.BDC mice treated with YTS 177/105 versus control 2A3 (*P ⁇ 0.05).
- FIG. 6A-6C 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 and 6C BDC2.5 CD4 + T cells injected into remission versus control NOD mice (FIG. 6B) fail to proliferate in the PLN.
- FIG. 6C Suppressed proliferation of injected BDC2.5 CD4 + T cells in remission NOD mice is overcome by deleting Foxp3 + Treg by administration of the anti-CD25 antibody clone PC61.
- FIG. 7A-7D The frequency of Foxp3 + Treg is increased in the PLN of long-term remission NOD mice (e.g., 200 days post-YTSl77/YTSl05) (FIG. 7 A, 7B).
- FIG. 7C The frequency of CD62L M expressing Foxp3 + Treg is increased in YTS177/YTS105 treated NOD mice.
- FIG. 7D CD62L 1 ’ 1 Foxp3 + Treg from the PLN but not the spleen of YTS177/YTS105 treated NOD mice exhibit increased secretion of IL-10 and TGF l measured by ELISA.
- FIG. 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 gp33-4l .
- FIG. 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 (*R ⁇ 10 2 ), and
- FIG. 9C serum IFNy measured 72 hours post-treatment (**R ⁇ 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).
- FIG. 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.
- 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 ⁇ 0.0l 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 anti-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.
- FIGS. 17A-B - Human CD4 Protein sequence (only the extracellular domain was used for array design (SEQ ID NO: 169).
- FIG. 17B Rendering of the CD4 homodimer as present in lcdh.pdb.
- FIG. 18 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 read- out. Constructs containing right amino acid sequence in the correct conformation best bind the antibody. (SEQ ID NO: 170)
- FIG. 19 - 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. 20 - The target protein contains a-helixes, b-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. 21 Box plot schematic.
- FIG. 22 Intensity profile and corresponding heatmap representation of a response from a polyclonal serum screened on a library of overlapping linear peptides.
- FIG. 23 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 etal ., (1978) The American Statistician, 32: 12-16).
- FIG. 24 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. 25 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 b- tum 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. 26 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 grey and background signals are plotted in black. (SEQ ID NOS: 171-197)
- FIGS. 27A-B - Human CD8 Protein sequence (only the extracellular domain was used for array design (SEQ ID NO: 198).
- FIG. 27B Rendering of the CD8 homodimer as present in lcdh.pdb.
- FIG. 28 Intensity profile and corresponding heatmap representation of a response from a polyclonal serum screened on a library of overlapping linear peptides.
- FIG. 29 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 etal. , (1978) The American Statistician, 32: 12-16).
- FIG. 30 Overlay of intensity profiles recorded for antibody CD8 mAb under low stringency conditions with linear (blue), single loop (purple), a-helical (green) and b-tum (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. 31 Heatmap representation of the intensity profile recorded for antibody
- FIG. 32 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 (SEQ ID NO: 167) and 109 NEGYYFCSA 117 (SEQ ID NO: 168) are shown in blue and red, respectively.
- FIG. 33 Molecular surface of the CD8 dimer with single subunits colored in white and grey. Peptide stretches 60 PRGAAASPTFLLY 72 (SEQ ID NO: 167) and 109 NEGYYFCSA 117 (SEQ ID NO: 168) are shown in blue and red, respectively.
- FIGS. 34A-B Nondepleting anti-CD4/CD8a antibody therapy prevents T cell- mediated multi-organ autoimmunity in NOD mice deficient of Aire expression. (FIG.
- FIG. 35 Tissues remain free of infiltration in NOD mice deficient of Aire expression following treatment with nondepleting anti-CD4/CD8a antibody. Representative H&E staining of various tissues of NOD mice lacking Aire expression and treated with 500 pg of YTS177 plus YTS105 at day 4 and day 6 post birth or left untreated (UTX).
- FIG. 36 Nondepleting anti-CD4 antibody therapy prevents and treats experimental autoimmune encephalomyelitis (EAE).
- EAE experimental autoimmune encephalomyelitis
- a peptide of proteolipid protein plus pertussis toxin 500 pg of YTS177 injected at different times, and clinical symptoms scored.
- Untreated mice served as controls. *r ⁇ 10 1 , vs d3 and dl 1 YTS injection (Student’s t test).
- FIG. 37 Nondepleting anti-huCD4/CD8a bispecific antibody has increased tissue-specific effects on huCD45 + CD3 + CD4 + and CD8 + T cells in humanized NRG-huPBL mice over co-therapy of anti-huCD4 plus anti-CD8a monospecific antibody cocktail.
- NRG-huPBL mice were treated with either anti-huCD4/CD8a bispecific antibody, a cocktail of anti-huCD4 plus anti-CD8a monospecific IgG4 or control IgG4, and CD4 + and CD8 + T cell numbers measured in the pancreas, liver and spleen 72 hrs post-treatment.
- FIG. 38 Residual pancreatic human CD4 + and CD8 + T cells exhibit a qualitatively distinct surface phenotype after coreceptor therapy.
- FIG. 39 Human T cell purging of the pancreas by coreceptor therapy is blocked by inhibiting SlPrl .
- FIG. 40 Expression of genes regulated by the Foxol transcription factor is increased by nondepleting anti-huCD4 and anti-huCD8a bispecific antibody.
- Humanized NRG-huPBL mice were treated with 2 mg of control IgG4 (Ig) or anti- huCD4/CD8a bispecific antibody for 24 hrs.
- CD4 + or CD8 + T cells were FACS- sorted from the pancreas.
- T cell RNA was isolated, and cDNA synthesized for qRT-PCR.
- Genes encoding CCR7, KLF2 and SlPrl are regulated by Foxol .
- Expression of CD3 is being used as a control. Data is presented as the delta-delta CT for T cells from bispecfic versus Ig antibody treated mice. *p ⁇ 0.05 (Student’s t test).
- FIG. 41 Nondepleting anti-huCD4/CD8a bispecific antibody suppresses activation and increases CD127 surface expression by human T cells in vivo.
- Humanized NRG-huPBL mice were treated with 2 mg of control IgG4 (Ig) or anti- huCD4/CD8a bispecific antibody for 72 hrs.
- Pancreatic T cells were then assessed via flow cytometry for expression of the activation marker CD69, and Foxol - regulated CD127. Representative histograms are provided.
- CD4/CD8 bispecific antibodies are provided herein which have been demonstrated to provide an effective treatment for autoimmune/inflammatory disease.
- an autoimmune disease is a condition arising from an abnormal immune response to a normal body part. A substantial minority of the population suffers from these diseases, which are often chronic, debilitating, and life-threatening.
- Autoimmune diseases have a wide variety of different effects. They do tend to have one of three characteristic pathological effects which characterize them as autoimmune diseases: damage to or destruction of tissues
- autoimmune diseases such as exposure to radiation or certain drugs which can damage tissues of the body. Infection can also be a trigger of some autoimmune diseases for example Lupus which is thought to be a milder version of an idiopathic disorder where there is an increased production of antihistone antibodies.
- the human immune system typically produces both T-cells and B-cells that are capable of being reactive with self-antigens, but these self-reactive cells are usually either killed prior to becoming active within the immune system, placed into a state of anergy (silently removed from their role within the immune system due to over-activation), or removed from their role within the immune system by regulatory cells. When any one of these mechanisms fail, it is possible to have a reservoir of self-reactive cells that become functional within the immune system.
- the mechanisms of preventing self-reactive T-cells from being created takes place through Negative selection process within the thymus as the T-cell is developing into a mature immune cell.
- Some infections such as Campylobacter jejuni , have antigens that are similar (but not identical) to human molecules.
- a normal immune response to C. jejuni can result in the production of antibodies that also react to a lesser degree with receptors on skeletal muscle (i.e., Myasthenia gravis).
- a major understanding of the underlying pathophysiology of autoimmune diseases has been the application of genome wide association scans that have identified a degree of genetic sharing among the autoimmune diseases.
- Autoimmunity is the presence of self-reactive immune response (e.g ., auto-antibodies, self- reactive T-cells), with or without damage or pathology resulting from it. This may be restricted to certain organs or involve a particular tissue in different places.
- autoimmune disorders include ankylosing spondylitis, arthritis, rheumatoid arthritis, osteoarthritis, Chagas disease, dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis disease, Hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, Idiopathic thrombocytopenic purpura, inflammatory bowel disease, Celiac's disease, Crohn's disease, eosinophilic gastroenteritis, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome, infective colitis, indeterminate colitis interstitial cystitis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus, neon
- autoimmune diseases are treated using anti-inflammatory drugs and biologies that impair the stimulation of the immune cells that cause the disease, or that block the effects of molecules produced by immune cells once stimulated.
- anti-inflammatory drugs and biologies that impair the stimulation of the immune cells that cause the disease, or that block the effects of molecules produced by immune cells once stimulated.
- agents include steroids, non-steroidal anti-inflammatory drugs, and antibodies that block immune receptors or immune effector molecules.
- an antibody as disclosed herein is targeted to CD4 and CD8 antigens.
- the antibody is a bispecific antibody targeted to both CD4 and CD8.
- 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.
- CD4 Cluster of differentiation 4 is a glycoprotein having a molecular weight of about
- 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.
- TCR T cell receptor
- CD4 is a type I transmembrane protein in which four immunoglobulin superfamily domains (designated in order as Dl 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.
- MHC major histocompatibility complex
- 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).
- HCV human immunodeficiency virus
- CD4-positive cells examples include CD4-positive T cells such as a Thl cell, a Th2 cell, a Thl7 cell, a regulatory T cell (Treg), and a gdT cell. Further, CD4-positive cells are associated with diseases including cancer and inflammatory diseases (e.g. , autoimmune disease or an allergic disease).
- CD4-positive T cells such as a Thl cell, a Th2 cell, a Thl7 cell, a regulatory T cell (Treg), and a gdT cell.
- 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.
- 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 et al, Blood, 109(11):4655-4662, 2007).
- 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 aa homodimer or as an ab 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 b 2 ih and the a2 and a3 domains of MHC Class I molecules using its b 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.
- IgSF Ig superfamily
- CD8a chain associated tyrosine protein kinase p56lck 4 ' 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 el al, J Exp Med, 189: 1521-30, 1999).
- 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).
- 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.
- TCR T-cell receptor
- anti-CD8 antibodies including monoclonal antibodies, known in the art, including: 2D2; 4D12.1; 7B12 IG1 1; 8E-1.7; 8G5; 14; 2lThy; 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; ClO; C12/D3; CD8-4C9; CLB-T8/1; CTAG-CD8, 3B5; F80-1D4D11; F101-87 (S-T8a); GlO-I; GlO-l .
- ET.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.
- the mechanism(s) by which CD4 mAbs produce these effects are still not clear.
- 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.
- CD4 (and CD8) mAbs could affect lymphocyte functions simply through binding to the antigen on the cell surface without cell lysis.
- 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.
- non-depleting CD4 and CD8 antibodies can also produce tolerance to foreign immunoglobulins, bone marrow and skin grafts.
- 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.
- the methods for generating monoclonal antibodies 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.
- 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).
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- 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.
- the immunogenicity of a particular immunogen composition can be enhanced by the use of non specific stimulators of the immune response, known as adjuvants.
- 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.
- 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.
- the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.
- 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.
- B lymphocytes B lymphocytes
- 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 el al. (1977).
- PEG polyethylene glycol
- 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 and 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.
- the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
- HAT medium a source of nucleotides
- 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.
- EBV Epstein Barr virus
- 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.
- HPRT hypoxanthine phosphoribosyl transferase
- 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.
- 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).
- the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
- pristane tetramethylpentadecane
- 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.
- human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant.
- 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.
- 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.
- RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector.
- 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 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/affmity 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.
- monoclonal antibodies having CDRs from the heavy and light chains as illustrated herein are provided. Such antibodies may be produced by the clones discussed below.
- the antibodies may be defined by their variable sequence, which include additional“framework” regions.
- the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below.
- 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.
- 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).
- antibodies may be defined by the activities associated herewith.
- the present antibodies are characterized as (a) non-depleting, (b) having the ability to reduce effector T cell number in the subject’s inflamed tissues including but not limited to the liver, pancreas, salivary glands, ovaries, testes, skin, central nervous system, synovial tissue, gastrointestinal tract, thyroid, kidneys, lungs or eyes, such as CD4+ or CD8+ T cells, and (c) having the ability to increase Fox3+ Treg activity in the subject’s inflamed tissues.
- 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.
- Recombinant full length IgG antibodies were generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies were collected an purified from the 293 or CHO cell supernatant.
- 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.
- 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.
- CMV cytomegalovirus
- SV40 Simian Virus 40
- AdMLP adenovirus major late promoter
- the recombinant expression vectors can also include origins of replication and selectable markers (see, e.g, U.S. Patent 4,399,216 and U.S. Patent 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.
- drugs such as G418, hygromycin or methotrexate
- the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate
- 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 dhfir- 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.
- 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.
- HPLC high performance liquid chromatography
- 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.
- 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.
- 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.
- 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).
- an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g, a chimeric, or CDR-grafted antibody).
- modifications such as introducing conservative changes into an antibody molecule.
- 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.
- 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).
- an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein.
- 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.
- 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.
- 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.
- a Single Chain Variable Fragment 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.
- 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 etal. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries.
- scFvs single-chain antibodies
- 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 10 6 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.
- scFv 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.
- the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.
- a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit.
- the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (/. 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.
- a stablizing and coagulating agent e.g., a stablizing and coagulating agent.
- dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created.
- 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.).
- primary amine group e.g, N-hydroxy succinimide
- a thiol group e.g, pyridyl disulfide, maleimides, halogens, etc.
- 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).
- 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.
- SMPT cross-linking reagent
- 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.
- thiolate anions such as glutathione which can be present in tissues and blood
- the SMPT cross-linking reagent 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.
- non-hindered linkers also can be employed in accordance herewith.
- Other useful cross-linkers 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.
- a wild-type IgG antibody contains two identical fragments termed "fragment, antigen binding” (or Fab), each of which is composed of the VH and CH1 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.
- fragment, antigen binding or Fab
- Fab fragment, antigen binding
- Each Fab directs binding of the antibody to the same antigen.
- fragment, antigen binding or Fab
- fragment, antigen binding or Fab
- bi-specific antibody 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.
- bi-specific antigen binding compound refers to Fab-Fab and IgG- Fab molecules.
- FIG. 1, included herein, provides a schematic diagram of the structure of bi- specific antibodies (IgG BsAb) as well as the Fab-Fab and IgG-Fab formats contemplated by the methods and compounds of the present disclosure.
- the methods and compounds 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.
- 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.
- 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.
- 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 (z.e., in the N-terminal direction) from the first amino acid in the reference HFR3.
- Design A of the present disclosure comprises the replacement of a WT amino acid in HCDR2 with a glutamic acid (E).
- amino acid residue X66 is the most N-terminal (first) amino acid residue of variable region heavy chain framework three (HFR3).
- HFR3 variable region heavy chain framework three
- 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.
- 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.
- 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.
- “Q39K” refers to a replacement of a glutamine at residue 39 with a lysine.
- 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 (mAh)), including any eukaryotic, prokaryotic, or phage clone.
- a compound of the present disclosure exists in a homogeneous or substantially homogeneous population.
- the antibody, Fab or other antigen binding compound, or a nucleic acid encoding the same is provided in "isolated” form.
- 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.
- 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.
- 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, NSO, DG-44, and CHO cells.
- 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.
- 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 hydroxyapatite chromatography. Recovered products may be immediately frozen, for example at -70 °C., or may be lyophilized.
- 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.
- 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 CH1 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 Rabat, and said heavy chain CH1 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 1
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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).
- 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 CH1 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 Rabat, and said heavy chain CH1 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 substitute
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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).
- 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 CH1 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 CH1 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
- 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 CH1 domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CH1 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
- 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 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 CH1 domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CH1 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;
- 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 CH1 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 Rabat, and said heavy chain CH1 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
- 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 CH1 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 CH1 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
- 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 CH1 domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CH1 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
- 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 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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).
- 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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),
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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 arginie 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 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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 arginie 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 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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
- 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 CH1 domain, wherein said first heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y), and said first IgG heavy chain constant CH1 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
- 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 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 CH1 domain, wherein said first heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y), and said first IgG heavy chain constant CH1 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 a first
- 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 Rabat, and a CH1 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 (176
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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 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 Rabat, and a CH1 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
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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 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 Rabat, and a CH1 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
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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 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 Rabat, and a CH1 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
- the present disclosure provides a method comprising one or more of the following: said first nucleic acid encodes a heavy chain CH1 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 CH1 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 (39
- 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 CH1 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 (39
- 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 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.
- 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 Rabat, a CH1 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
- 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.
- the present disclosure provides any of the Fabs, bispecific antibodies, nucleic acids or host cells as exemplified herein.
- 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 CH1 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
- 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.
- 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-polypeptide 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.
- 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.
- polypeptide 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.
- affinity column which binds to a tagged portion of the polypeptide.
- complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody.
- agents i.e., protein A
- 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 amounts 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.
- 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.
- pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the ET.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
- 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.
- 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.
- compositions 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 generally will involve the use of intravenous or intramuscular inj ections.
- the forms of antibody can be of any source, but in particular as high-titer humanized monoclonal antibodies (MAb).
- MAb monoclonal antibodies
- Such immunity generally lasts for a certain period of time, and further administration may be required.
- passive immunity provides immediate protection.
- the antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.
- 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.
- a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- 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.
- bispecific antibody peptide therapy can be used successfully in conjunction with another therapeutic or regimen to treat the disease.
- These therapies would be provided in a combined amount effective to address one or more symptom or underlying cause of disease.
- 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 bispecific antibody of the present disclosure and the other therapy.
- one treatment may precede or follow the other therapy by intervals ranging from minutes to weeks.
- 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.
- bispecific antibody is“A” and the other therapy is“B,” as exemplified below:
- both therapies are delivered to a subject in a combined amount effective to achieve that goal.
- Specific combination therapy strategies include combining our bispecific antibody therapy with the following classes of immunosuppressive/ anti - inflammatory drugs: immunosuppressive biologies (including antibodies), steroids, cytostatics, and/or drugs acting on immunophilins.
- immunosuppressive drugs examples include: dexamethasone, hydrocortisone, methylprednisone, prednisone, budesonide, prednisolone, methotrexate, azathioprine, leflunomide, mycophenolate, cyclosporine, tacrolimus, sirolimus, everolimus, abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab, muromonab, myriocin or fmgolimod. V. Kits
- 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.
- Induction of remission requires both YTS177 and YTS105 to induce efficient remission.
- Treatment of newly diabetic NOD mice with 800 ug of YTS105 (FIG. 2A) or YTS177 (FIG. 2B) alone has no or only a limited effect on blood glucose levels and diabetes reversal.
- FIG. 3A 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 ug of YTS177/YTS105 versus isotype control 2A3 antibody (FIG. 3 A).
- immunoregulatory Foxp3 + Treg are also reduced in the pancreas and PLN but not the spleen of YTSl77/YTSl05-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 YTS177/YTS105 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).
- qRT-PCR quantitative reverse transcriptase-polymerase chain reaction
- Downregulation of mRNA encoding proinflammatory cytokines and chemokines is detected as early as 6 hours post-YTSl77/YTSl05 treatment (FIG. 4B). In contrast, significant T cell purging is detected at 18 hours post-treatment.
- YTSl77/YTSl05-induced T cell purging is due to enhanced reactivity to sphingosine-l -phosphate (S1P), a major regulator of T cell tissue egress.
- S1P sphingosine-l -phosphate
- NOD.BDC BDC2.5 clonotypic T cell receptor
- 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 S1P measured by flow cytometry.
- CD4+ T cells from the PLN but not the spleen of YTS177 treated NOD.BDC mice exhibit enhanced chemotaxis to S1P (FIG. 5B).
- FIG. 8A lymphocytic choriomeningitis virus (LCMV) challenge was tested.
- NOD mice were inoculated i.p. with 1,000 viral particles of LCMV (Armstrong) and then treated 72 hours later with 500 ug of YTS177/YTS105 or control 2A3.
- FIG. 8B an equivalent frequency and number (FIG. 8C) of LCMV- specific CD8 + T cells, measured by flow cytometric detection of H2Db tetramers complexed with the LCMV gp33-4l peptide, was seen. Therefore, coreceptor therapy has a minimal effect on T cell reactivity to a viral pathogen.
- NOD mice lacking expression of the transcription factor AIRE were employed.
- NOD. AIRE mice develop systemic T cell- mediated autoimmunity, in which multiple tissues are infiltrated by T cells and destroyed. The culmination of this tissue-destruction results in significant wasting in NOD.
- AIRE 7 mice At 4 and 6-days post-birth, NOD. AIRE 7 neonates received YTS177/YTS105 or were left untreated, and wasting disease monitored. As expected, all untreated NOD.
- FIG. 34A AIRE 7 mice exhibited significant weight-loss and wasting (FIG. 34A). In contrast, the majority of YTS177/YTS105- treated animals remained free of the wasting disease (FIG. 34A). This protection from disease corresponded with markedly reduced T cell infiltration of various tissues such as the exocrine pancreas, salivary gland, liver, ovaries and testes (FIGS. 34B and 35). Importantly, these results demonstrate the coreceptor therapy is highly effective at modulating autoimmunity, regardless of the tissues being targeted by T cells. Therefore, it is expected that coreceptor will be effective for most, if not all, T cell-mediated autoimmune diseases and pathologies.
- a murine EAE model of Multiple Sclerosis was employed.
- SJL mice were immunized with a peptide derived from the autoantigen proteolipid protein (PLP) to induce EAE.
- PLP autoantigen proteolipid protein
- SJL mice were treated twice with 500ug of YTS177.
- This model of EAE is driven by PLP-specific CD4 + T cells; CD8 + T cells have no role in disease initiation of progression.
- NRG-huCD8a IgG4 To test the in vivo effects of engineered nondepleting anti-huCD4 and - 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.
- PBMC peripheral blood mononuclear cells
- xGVHD xenogeneic graft versus host disease
- NRG-huPBL mice after huT cell reconstitution with nondepleting anti-huCD4 and -huCD8a IgG4 delays xGVHD monitored in part via a weight loss (FIG. 9B).
- coreceptor therapy reduces serum levels of human proinflammatory cytokines such as IFNy (FIG. 9C) measured by ELISA, and induces CD4+ and CD8+ 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).
- 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).
- FIG. 12A flow cytometric binding data is provided for the bispecific HC8.4 which has been engineered with the YTS 177 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).
- the nondepleting anti-huCD4/huCD8a bispecific antibody exhibits enhanced tissue-specific purging properties in vivo over the addition of a cocktail of huCD4 and huCD8a monospecific antibodies.
- huT cell purging is increased in the pancreas and liver of NRG-huPBL mice within 72 hours of receiving anti-huCD4/huCD8a bi specific antibody versus the combination of the monospecific anti-huCD4 and -huCD8a IgG4 (FIG. 37).
- 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. 37).
- 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 and 16).
- Sphingosine-l -phosphate is a major regulator of tissue egress by T cells.
- FTY720 This drug blocks the function of the receptor for S1P; namely SlPrl .
- NRG-huPBL mice received the cocktail of 500 pg of monospecific anti-huCD4 and -huCD8a IgG4 plus/minus 10 mg/kg of FTY720.
- T cell numbers were markedly reduced in NRG- huPBL mice treated with anti-huCD4 and -huCD8a IgG4 versus control IgG4 (FIG. 39).
- T cell-mediated purging of the pancreas by anti-huCD4 and -huCD8a IgG4 was blocked by administration of FTY720 (FIG. 39).
- gene expression of SlPrl is regulated by the Foxol/KLF2 transcription pathway.
- NRG-huPBL mice were treated with the cocktail of monospecific anti-huCD4 and anti-huCD8a IgG4 or control IgG4.
- CD4 + and CD8 + T cells were FACS-sorted from the pancreas.
- mRNA levels encoding KLF2, SlPrl and CCR7, another member of the transcription pathway were then measured by qRT-PCR.
- Example 2 CD4 Epitope Mapping (Methods) SYNTHESIS OF PEPTIDES.
- 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 A-hydroxybenzotri azole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA).
- BocHMDA tbutyloxycarbonyl-hexamethylenediamine
- DCC dicyclohexylcarbodiimide
- HOBt A-hydroxybenzotri azole
- 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.
- 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)).
- 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 pl wells).
- the peptide arrays are gently shaken in the solution for 30 to 60 minutes while completely covered in solution.
- 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 H 2 0 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 (overnight 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 pl/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.
- CCD charge coupled device
- 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.
- 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. 21). 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 depict a graphical representation of the intensity profile recorded for a given sample.
- 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. 22 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. 21) and a heatmap (bottom panel FIG. 22). 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).
- Looped peptides of length 17 On positions 2 - 16 are l5-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”).
- CTVELT2TASQKKSIQC (SEQ ID NO: 62)
- CELT2TASQKKSIQFHC (SEQ ID NO: 63)
- CTASQKKSIQFHWKNSC (SEQ ID NO: 65)
- CSQKKSIQFHWKNSNQC (SEQ ID NO: 66)
- 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 a-helical structure. Native Cys are replaced by Cys- acm (denoted“2”).
- CADSCRSLWDQGNFPLIIKNLK (SEQ ID NO: 75)
- CDQGCFPLIIKNLKIEDSDTYI SEQ ID NO: 76
- b-tum epitope mimics of length of length 22.
- 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 b-tum formation. Cys are inserted on positions 1 and 22 and joined by means of mP2 CLIPS to stabilize the b-turn structure. Native Cys are replaced by Cys- acm (denoted“2”).
- CVVLGKKGDTPGLT2TASQKKC (SEQ ID NO: 79)
- CVLGKKGDTVPGT2TASQKKSC (SEQ ID NO: 80)
- CDTVELT2TAPGKKSIQFHWKC (SEQ ID NO: 86)
- 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”).
- 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).
- 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).
- 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. 18.
- 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. 19). 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 b-strands. A schematic of this approach is drawn in FIG. 20, where a single loop element is mimicked using mP2 CLIPS chemistry.
- FIG. 23 A graphical overview of the complete dataset is given in FIG. 23. 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. 24-26.
- CLIPS Chemically Linked Peptides on Scaffolds
- 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 pl wells).
- the peptide arrays are gently shaken in the solution for 30 to 60 minutes while completely covered in solution.
- the peptide arrays are washed extensively with excess of H 2 0 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 (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (SB A; Table 4) for one hour at 25°C. After washing, the peroxidase substrate 2,2’-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 pl/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.
- CCD charge coupled device
- 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.
- 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. 21). The main application of this boxplot is in finding the baseline levels and estimating signal over noise to allow data quality assessment.
- top panel in FIG. 28 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. 28 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. 28) and a heatmap (bottom panel FIG. 28). 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).
- RV SPLDRTWNLGET V (SEQ ID NO: 100)
- V SPLDRTWNLGET VE (SEQ ID NO: 101)
- SPLDRTWNLGET VEL SEQ ID NO: 102
- VLLSNPTSG2SWLFQ (SEQ ID NO: 115)
- Looped peptides of length 17 On positions 2 - 16 are l5-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”).
- CDRTWNLGETVELK2QC (SEQ ID NO: 131)
- CTWNLGETVELK2QVLC SEQ ID NO: 132
- CTVELK2QVLLSNPTSC SEQ ID NO: 1357
- CELK2QVLLSNPTSG2C (SEQ ID NO: 136)
- 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”).
- CDFRCENEGYYF2SALSNSIMY (SEQ ID NO: 142)
- b-tum epitope mimics of length of length 22.
- 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 b-tum formation. Cys are inserted on positions 1 and 22 and joined by means of mP2 CLIPS to stabilize the b-turn structure. Native Cys are replaced by Cys- acm (denoted“2”).
- 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”).
- 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).
- 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. 19). 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 b-strands. A schematic of this approach is drawn in FIG. 20, where a single loop element is mimicked using mP2 CLIPS chemistry.
- FIG. 29 A graphical overview of the complete dataset is given in FIG. 29. 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.
- 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 (SEQ ID NO: 168) in three different peptide sets - linear, b-tum and disulfide bridges.
- 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
Abstract
The present disclosure is directed to bispecific antibodies binding to CD4 and CDS, and their use in treating autoimmune/inflammatory diseases. Specifically, the disclosure provides methods of treating a T cell mediated autoimmune/inflammatory disease in a subject comprising providing a bispecific antibody or antibody fragment having binding specificity for CD4 and CDS to said subject, wherein said autoimmune/inflammatory disease is not diabetes. Further disclosed are complementary determining regions (CDRs) sequences of bispecific antibodies.
Description
DESCRIPTION
USE OF A CD4/CD8 BISPECIFIC ANTIBODY FOR THE TREATMENT OF
AUTOIMMUNE/INFLAMMATORY DISORDERS
PRIORITY CLAIM
This application claims benefit of priority to U.S. Provisional Application Serial No. 62/732,545, filed September 17, 2018, the entire contents of which are hereby incorporated by reference.
GOVERNMENT SUPPORT CLAUSE
This invention was made with government support under Grant Numbers DK100256 and All 15752 awarded by the National Institutes of Health. The government has certain rights in the invention.
INCORPORATION OF SEQUENCE LISTING
The sequence listing that is contained in the file named“DURAP0005WO_ST25.txt”, which is 82 KB (as measured in Microsoft Windows®) and was created on September 16, 2019, 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
An autoimmune disease is a condition arising from an abnormal immune response to a normal body part, often starting in young adulthood. There are at least 80 types of autoimmune diseases. Nearly any body part can be involved. Common symptoms include low grade fever and feeling tired. Often symptoms come and go. About 24 million (7%) people in the United States are affected by an autoimmune disease. Women are more commonly affected than men, and it has been estimated that autoimmune diseases are among the leading causes of death among women in the United States in all age groups up to 65 years.
The cause is generally unknown. Some autoimmune diseases such as lupus run in families, and certain cases may be triggered by infections or other environmental factors. Some
common autoimmune diseases include celiac disease, diabetes mellitus type 1, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. The diagnosis can be difficult to determine given the similar and overlapping symptoms, and the distinguishing causative factors for each of these diseases are still being sorted out.
Treatment depends on the type and severity of the condition. Nonsteroidal anti inflammatory drugs (NSAIDs) and immunosuppressants are often used. Intravenous Immunoglobulin may also occasionally be used. While treatment usually improves symptoms, they do not typically cure the disease. A further challenge is being able to tailor a response to the particular aspects of an individual’s disease. Thus, a therapeutic approach that was valid for all subject’s with a given autoimmune disease would be extremely valuable, and an approach that was valid for multiple clinically distinct autoimmune diseases would be a remarkable advance in the care of patients afflicted with these maladies.
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.
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 (SEQ ID NO: 168) and/or PRGAAASPTFLLY (SEQ ID NO: 167). 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 pattern, 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 an autoimmune/inflammatory disease 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 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 any of the subject’s inflamed tissues including but not limited to the liver, pancreas, salivary glands, ovaries, testes, skin, central nervous system, synovial tissue, gastrointestinal tract, thyroid, kidneys, lungs or eyes, such as CD4+ or CD8+ T cells. In certain aspects, providing results in an increase in immunosuppressive function of Fox3+ Treg cells in the subject’s inflamed tissues.
In certain aspects, the method further comprises administering to said subject a second autoimmune/anti-inflammatory 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, gd+ T cells, NK T cells, CD4+ T cells and CD8+ T cells. CD4+ T cells include THO, THI and TH2 cells, as well as regulatory T cells (Treg). There are at least three types of regulatory T cells: CD4+ CD25+ Treg, CD25 TH3 Treg, and CD25 TRT Treg.“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 preferred 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 mAh, 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. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 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. 1B) 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.
FIG. 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 (h=10).
FIG. 3A-3B - Induction of remission correlates with T cell purging of the pancreas. (FIG. 3 A) 6 days post-YTSl77/YTSl05 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 2A3 treated NOD mice.
FIG. 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 2 A3 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.
FIG. 5A-5B - CD4 crosslinking of NOD.BDC PLN but not splenic T cells increases sphingosine-l -phosphate (S1P) chemotaxis. (FIG. 5 A) Schematic of experimental approach. (FIG. 5B) Percent migration to S1P in an in vitro transwell assay of CD4+ T cells from the spleen or PLN of NOD.BDC mice treated with YTS 177/105 versus control 2A3 (*P<0.05).
FIG. 6A-6C - 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 and 6C) BDC2.5 CD4+ T cells injected into remission versus control NOD mice (FIG. 6B) fail to proliferate in the PLN. (FIG. 6C) Suppressed proliferation of injected BDC2.5 CD4+ T cells in remission NOD mice is overcome by deleting Foxp3+Treg by administration of the anti-CD25 antibody clone PC61.
FIG. 7A-7D - The frequency of Foxp3+Treg is increased in the PLN of long-term remission NOD mice (e.g., 200 days post-YTSl77/YTSl05) (FIG. 7 A, 7B). (Fig 7C) The frequency of CD62LM expressing Foxp3+Treg is increased in YTS177/YTS105 treated NOD mice. (FIG. 7D) CD62L1’1 Foxp3+Treg from the PLN but not the spleen of YTS177/YTS105 treated NOD mice exhibit increased secretion of IL-10 and TGF l measured by ELISA.
FIG. 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 gp33-4l .
FIG. 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 (*R<10 2), and (FIG. 9C) serum IFNy measured 72 hours post-treatment (**R<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). FIG. 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<0.0l 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 anti-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.
FIGS. 17A-B - (FIG. 17 A) Human CD4 Protein sequence (only the extracellular domain was used for array design (SEQ ID NO: 169). (FIG. 17B) Rendering of the CD4 homodimer as present in lcdh.pdb.
FIG. 18 - 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 read- out. Constructs containing right amino acid sequence in the correct conformation best bind the antibody. (SEQ ID NO: 170)
FIG. 19 - 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. 20 - The target protein contains a-helixes, b-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. 21 - Box plot schematic. Source: flowingdata.com
FIG. 22 - Intensity profile and corresponding heatmap representation of a response from a polyclonal serum screened on a library of overlapping linear peptides.
FIG. 23 - 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 etal ., (1978) The American Statistician, 32: 12-16).
FIG. 24 - 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. 25 - 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 b- tum 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. 26 - 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 grey and background signals are plotted in black. (SEQ ID NOS: 171-197)
FIGS. 27A-B - (FIG. 27 A) Human CD8 Protein sequence (only the extracellular domain was used for array design (SEQ ID NO: 198). (FIG. 27B) Rendering of the CD8 homodimer as present in lcdh.pdb.
FIG. 28. Intensity profile and corresponding heatmap representation of a response from a polyclonal serum screened on a library of overlapping linear peptides.
FIG. 29. 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 etal. , (1978) The American Statistician, 32: 12-16).
FIG. 30. Overlay of intensity profiles recorded for antibody CD8 mAb under low stringency conditions with linear (blue), single loop (purple), a-helical (green) and b-tum (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. 31. 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 grey and background signals are plotted in black. (SEQ ID NOS: 199-208)
FIG. 32. 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 (SEQ ID NO: 167) and 109 NEGYYFCSA 117 (SEQ ID NO: 168) are shown in blue and red, respectively.
FIG. 33. Molecular surface of the CD8 dimer with single subunits colored in white and grey. Peptide stretches 60 PRGAAASPTFLLY 72 (SEQ ID NO: 167) and 109 NEGYYFCSA 117 (SEQ ID NO: 168) are shown in blue and red, respectively. FIGS. 34A-B. Nondepleting anti-CD4/CD8a antibody therapy prevents T cell- mediated multi-organ autoimmunity in NOD mice deficient of Aire expression. (FIG. 34A) NOD mice lacking Aire expression (AIRE ) were treated with 500ug of both anti-CD4 (YTS177) and anti-CD8 (YTS105) (n=l7) at day 4 and day 6 post birth or left untreated (UTX; n=l6) and monitored for disease. (FIG. 34B) Organs were taken from these mice and stained with H&E and infiltration quantitated via microscopy. *p<0.05 (Student’s t test).
FIG. 35. Tissues remain free of infiltration in NOD mice deficient of Aire expression following treatment with nondepleting anti-CD4/CD8a antibody. Representative H&E staining of various tissues of NOD mice lacking Aire expression and treated with 500 pg of YTS177 plus YTS105 at day 4 and day 6 post birth or left untreated (UTX).
FIG. 36. Nondepleting anti-CD4 antibody therapy prevents and treats experimental autoimmune encephalomyelitis (EAE). EAE was induced in SJL mice (n=5) with a peptide of proteolipid protein plus pertussis toxin, 500 pg of YTS177 injected at different times, and clinical symptoms scored. Untreated mice served as controls. *r<10 1, vs d3 and dl 1 YTS injection (Student’s t test).
FIG. 37. Nondepleting anti-huCD4/CD8a bispecific antibody has increased tissue-specific effects on huCD45+CD3+ CD4+ and CD8+ T cells in humanized NRG-huPBL mice over co-therapy of anti-huCD4 plus anti-CD8a monospecific antibody cocktail. NRG-huPBL mice were treated with either anti-huCD4/CD8a bispecific antibody, a cocktail of anti-huCD4 plus anti-CD8a monospecific IgG4 or control IgG4, and CD4+ and CD8+ T cell numbers measured in the pancreas, liver and spleen 72 hrs post-treatment.
FIG. 38. Residual pancreatic human CD4+ and CD8+ T cells exhibit a qualitatively distinct surface phenotype after coreceptor therapy. Representative flow cytometric histograms of human CD4+ and CD8+ T cells from the pancreas of
humanized NRG-huPBL mice 72 hrs after treatment with PBS or 500 pg of both anti-huCD4 and anti-huCD8 monospecific IgG4.
FIG. 39. Human T cell purging of the pancreas by coreceptor therapy is blocked by inhibiting SlPrl . Humanized NRG-huPBL mice were treated daily with lOmg/kg FTY720 from day 0 to 3. On day 1 mice were then treated with lmg control IgG4 or 500 pg of both anti-huCD4 and -huCD8a IgG4. 72 hrs post antibody treatment, T cells were enumerated in the pancreas via flow cytometry. Each data point is the average of 1-3 mice per donor. Each donor (n=5) is represented by a distinct symbol. *p<0.05 (two-way ANOVA).
FIG. 40. Expression of genes regulated by the Foxol transcription factor is increased by nondepleting anti-huCD4 and anti-huCD8a bispecific antibody. Humanized NRG-huPBL mice were treated with 2 mg of control IgG4 (Ig) or anti- huCD4/CD8a bispecific antibody for 24 hrs. CD4+ or CD8+ T cells were FACS- sorted from the pancreas. T cell RNA was isolated, and cDNA synthesized for qRT-PCR. Genes encoding CCR7, KLF2 and SlPrl are regulated by Foxol . Expression of CD3 is being used as a control. Data is presented as the delta-delta CT for T cells from bispecfic versus Ig antibody treated mice. *p<0.05 (Student’s t test).
FIG. 41. Nondepleting anti-huCD4/CD8a bispecific antibody suppresses activation and increases CD127 surface expression by human T cells in vivo. Humanized NRG-huPBL mice were treated with 2 mg of control IgG4 (Ig) or anti- huCD4/CD8a bispecific antibody for 72 hrs. Pancreatic T cells were then assessed via flow cytometry for expression of the activation marker CD69, and Foxol - regulated CD127. Representative histograms are provided.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
As discussed above, there remains a great need for improved therapies for autoimmune/inflammatory disease. Accordingly, CD4/CD8 bispecific antibodies are provided herein which have been demonstrated to provide an effective treatment for autoimmune/inflammatory disease. These and other aspects of the disclosure are described in detail below.
I. Autoimmune/Inflammatory Diseases
As discussed above, an autoimmune disease is a condition arising from an abnormal immune response to a normal body part. A substantial minority of the population suffers from these diseases, which are often chronic, debilitating, and life-threatening.
For a disease to be regarded as an autoimmune disease it needs to answer to Witebsky’s postulates:
direct evidence from transfer of disease-causing antibody or disease-causing T lymphocyte white blood cells;
indirect evidence based on reproduction of the autoimmune disease in experimental animals;
circumstantial evidence from clinical clues;
genetic evidence suggesting“clustering” with other autoimmune diseases;
autoimmune diseases are incurable
Autoimmune diseases have a wide variety of different effects. They do tend to have one of three characteristic pathological effects which characterize them as autoimmune diseases: damage to or destruction of tissues
altered organ growth
altered organ function
There are more than 80 illnesses caused by autoimmunity.
Autoimmune diseases affect approximately 2-5% of the western world's population.
Women are found to be more commonly affected than men. Environmental events can trigger some cases of autoimmune diseases such as exposure to radiation or certain drugs which can damage tissues of the body. Infection can also be a trigger of some autoimmune diseases for example Lupus which is thought to be a milder version of an idiopathic disorder where there is an increased production of antihistone antibodies.
The human immune system typically produces both T-cells and B-cells that are capable of being reactive with self-antigens, but these self-reactive cells are usually either killed prior to becoming active within the immune system, placed into a state of anergy (silently removed from their role within the immune system due to over-activation), or removed from their role within the immune system by regulatory cells. When any one of these mechanisms fail, it is possible to have a reservoir of self-reactive cells that become functional within the immune system. The mechanisms of preventing self-reactive T-cells from being created takes place through Negative selection process within the thymus as the T-cell is developing into a mature immune cell.
Some infections, such as Campylobacter jejuni , have antigens that are similar (but not identical) to human molecules. In this case, a normal immune response to C. jejuni can result in the production of antibodies that also react to a lesser degree with receptors on skeletal muscle (i.e., Myasthenia gravis). A major understanding of the underlying pathophysiology of autoimmune diseases has been the application of genome wide association scans that have identified a degree of genetic sharing among the autoimmune diseases. Autoimmunity, on the other hand, is the presence of self-reactive immune response ( e.g ., auto-antibodies, self- reactive T-cells), with or without damage or pathology resulting from it. This may be restricted to certain organs or involve a particular tissue in different places.
Some examples of autoimmune disorders include ankylosing spondylitis, arthritis, rheumatoid arthritis, osteoarthritis, Chagas disease, dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis disease, Hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, Idiopathic thrombocytopenic purpura, inflammatory bowel disease, Celiac's disease, Crohn's disease, eosinophilic gastroenteritis, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome, infective colitis, indeterminate colitis interstitial cystitis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus, neonatal lupus, mixed connective tissue disease, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, scleroderma, Sjogren's syndrome, Stiff person syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, alopecia areata, sarcoidosis, Addison's disease, or autoimmune hemolytic anemia.
In general, autoimmune diseases are treated using anti-inflammatory drugs and biologies that impair the stimulation of the immune cells that cause the disease, or that block the effects of molecules produced by immune cells once stimulated. Such agents include steroids, non-steroidal anti-inflammatory drugs, and antibodies that block immune receptors or immune effector molecules.
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 Dl 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 gdT 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 et al, 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 aa homodimer or as an ab 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 b2ih and the a2 and a3 domains of MHC Class I molecules using its b 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 p56lck4'5 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 el 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; 2lThy; 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; ClO; C12/D3; CD8-4C9; CLB-T8/1; CTAG-CD8, 3B5; F80-1D4D11; F101-87 (S-T8a); GlO-I;
GlO-l . l; HI208; HI209; HI212; HIT8a; HIT 8b; 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 /2lthy2D3; 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
ET.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 non specific 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 el 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 and 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 supernatant. 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/affmity 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 effector T cell number in the subject’s inflamed tissues including but not limited to the liver, pancreas, salivary glands, ovaries, testes, skin, central nervous system, synovial tissue, gastrointestinal tract, thyroid, kidneys, lungs or eyes, such as CD4+ or CD8+ T cells, and (c) having the ability to increase Fox3+ Treg activity in the subject’s inflamed tissues.
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.
Recombinant full length IgG antibodies were generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies were collected an purified from the 293 or CHO cell supernatant.
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. Patent 5,168,062 , U.S. Patent 4,510,245 and U.S. Patent 4,968,615. The recombinant expression vectors can also include origins of replication and selectable markers (see, e.g, U.S. Patent 4,399,216 and U.S. Patent 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 dhfir- 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 etal. (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 (/. 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 CH1 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 CH1 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 CH1 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 mis-matching 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. FIG. 1, included herein, provides a schematic diagram of the structure of bi- specific antibodies (IgG BsAb) as well as the Fab-Fab and IgG-Fab formats contemplated by the methods and compounds of the present disclosure.
The methods and compounds 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 (z.e., VH) amino acid residues (z.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 CH1 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 (z.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 (mAh)), 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, NSO, 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 hydroxyapatite 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 CH1 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 Rabat, and said heavy chain CH1 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 CH1 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 CH1 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 Rabat, and said heavy chain CH1 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 (135 Y) 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 CH1 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 CH1 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 CH1 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 CH1 domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CH1 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 CH1 domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CH1 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 CH1 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 Rabat, and said heavy chain CH1 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 CH1 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 CH1 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 CH1 domain, wherein said heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said heavy chain CH1 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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 CH1 domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CH1 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 CH1 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 CH1 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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 CH1 domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CH1 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 CH1 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 CH1 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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 CH1 domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CH1 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 CH1 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 CH1 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 arginie 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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 CH1 domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CH1 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 CH1 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 CH1 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 arginie 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 CH1 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 Rabat, and said first IgG heavy chain constant CH1 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 CH1 domain, wherein said second heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y) and said second IgG heavy chain constant CH1 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 CH1 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 CH1 domain, wherein said first heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y), and said first IgG heavy chain constant CH1 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 CH1 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 Rabat and said second IgG heavy chain constant CH1 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 CH1 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 CH1 domain, wherein said first heavy chain variable domain comprises a tyrosine substituted at residue 39 (39Y), and said first IgG heavy chain constant CH1 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 CH1 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 Rabat and said second IgG heavy chain constant CH1 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 CH1 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 Rabat, and a CH1 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 CH1 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 CH1 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 Rabat, and a CH1 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 CH1 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 CH1 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 Rabat, and a CH1 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 CH1 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 CH1 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 Rabat, and a CH1 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 CH1 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 CH1 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 CH1 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 Rabat 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 CH1 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 Rabat 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 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 Rabat, a CH1 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 CH1 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 CH1 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 CH1 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-polypeptide 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 amounts 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 Autoimmune/Inflammatory
Disorders
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 ET.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 inj ections. 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 required. 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 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 therapy.
These therapies would be provided in a combined amount effective to address one or more symptom or underlying cause of disease. 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 bispecific antibody of the present disclosure and the other 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. Specific combination therapy strategies include combining our bispecific antibody therapy with the following classes of immunosuppressive/ anti - inflammatory drugs: immunosuppressive biologies (including antibodies), steroids, cytostatics, and/or drugs acting on immunophilins. Examples of these immunosuppressive drugs to be using in combination therapies alongside our bispecific antibody therapy include: dexamethasone, hydrocortisone, methylprednisone, prednisone, budesonide, prednisolone, methotrexate, azathioprine, leflunomide,
mycophenolate, cyclosporine, tacrolimus, sirolimus, everolimus, abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab, muromonab, myriocin or fmgolimod. 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 T cell-mediated autoimmune disease
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 ug over 72 hours of each antibody within 10 days of diagnosis. YTS177/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. 1B). 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 YTS177 and YTS105 to induce efficient remission. Treatment of newly diabetic NOD mice with 800 ug of YTS105 (FIG. 2A) or YTS177 (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 ug 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 YTSl77/YTSl05-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 YTS177/YTS105 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-YTSl77/YTSl05 treatment (FIG. 4B). In contrast, significant T cell purging is detected at 18 hours post-treatment. YTSl77/YTSl05-induced T cell purging is due to enhanced reactivity to sphingosine-l -phosphate (S1P), 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 S1P measured by flow cytometry. CD4+ T cells from the PLN but not the spleen of YTS177 treated NOD.BDC mice exhibit enhanced chemotaxis to S1P (FIG. 5B).
To determine a role for immunoregulation in YTSl77/YTSl05-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). Suppressed proliferation of injected BDC2.5 CD4+ T cells in remission NOD mice is overcome
by deleting Foxp3+Treg by administration of the anti-CD25 antibody clone PC61 (FIG. 6C). This result indicates the critical role of Foxp3+Tregs in suppressing T cell proliferation. 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. 7A-C). These results are consistent with long-term protection being mediated by immunoregulation. Additionally, CD62Lhl Foxp3+Treg from the PLN but not the Spleen of long-term remission animals have increased secretion of the anti-inflammatory/immunosuppressive cytokines, IL- 10 and TGFp i (FIG. 7D), further implicating their role in long-term protection.
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. 8A). NOD mice were inoculated i.p. with 1,000 viral particles of LCMV (Armstrong) and then treated 72 hours later with 500 ug 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 gp33-4l peptide, was seen. Therefore, coreceptor therapy has a minimal effect on T cell reactivity to a viral pathogen.
ASSESSING THERAPEUTIC EFFICACY OF CORECEPTOR THERAPY IN NOD MICE LACKING AIRE EXPRESSION. To test the efficacy of coreceptor therapy under highly stringent conditions, NOD mice lacking expression of the transcription factor AIRE (NOD. AIRE ) were employed. Notably, NOD. AIRE mice develop systemic T cell- mediated autoimmunity, in which multiple tissues are infiltrated by T cells and destroyed. The culmination of this tissue-destruction results in significant wasting in NOD. AIRE 7 mice. At 4 and 6-days post-birth, NOD. AIRE 7 neonates received YTS177/YTS105 or were left untreated, and wasting disease monitored. As expected, all untreated NOD. AIRE 7 mice exhibited significant weight-loss and wasting (FIG. 34A). In contrast, the majority of YTS177/YTS105- treated animals remained free of the wasting disease (FIG. 34A). This protection from disease corresponded with markedly reduced T cell infiltration of various tissues such as the exocrine pancreas, salivary gland, liver, ovaries and testes (FIGS. 34B and 35). Importantly, these results demonstrate the coreceptor therapy is highly effective at modulating autoimmunity, regardless of the tissues being targeted by T cells. Therefore, it is expected that coreceptor will be effective for most, if not all, T cell-mediated autoimmune diseases and pathologies.
ASSESSING THERAPEUTIC EFFICACY OF CORECEPTOR THERAPY IN THE EAE MODEL OF MULTIPLE SCLEROSIS. As noted above, coreceptor therapy is
expected to prevent and/or treat T cell-mediated autoimmunity independent of the tissue being targeted. To further test this, a murine EAE model of Multiple Sclerosis (MS) was employed. Here, SJL mice were immunized with a peptide derived from the autoantigen proteolipid protein (PLP) to induce EAE. At different times during disease progression, SJL mice were treated twice with 500ug of YTS177. This model of EAE is driven by PLP-specific CD4+ T cells; CD8+ T cells have no role in disease initiation of progression. Administration of YTS177 at day 3 and 5 post-PLP-injection, a time prior to detection of clinical onset, resulted in all mice remaining free of EAE (FIG. 36). Strikingly, YTS177-treatment at days 11 and 13, when clinical symptoms are detected, resulted in remission of EAE (FIG. 36). These results demonstrate that coreceptor therapy is also effective at preventing and treating autoimmunity of the central nervous system and the brain. It is important to note that MS in patients is mediated by both CD4+ and CD8+ T cells. Therefore, the use of an anti-human (hu)CD4/CD8a bispecific antibody is both needed and expected to be therapeutically effective to prevent and/or treat MS.
CHARACTERIZATION OF NONDEPLETING ANTI-HUMAN (hu)CD4 AND
-huCD8a IgG4. To test the in vivo effects of engineered nondepleting anti-huCD4 and - 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 -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 CD8+ 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 YTS 177 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 -huCD8a IgG4 to huCD4+ and huCD8+ T cells, respectively, is also detected (FIG 14).
ASSESSING THERAPEUTIC EFFICACY OF CORECEPTOR THERAPY IN A HUMANIZED MODEL OF T CELL MEDIATED AUTOIMMUNITY. The nondepleting anti-huCD4/huCD8a bispecific antibody exhibits enhanced tissue-specific purging properties in vivo over the addition of a cocktail of huCD4 and huCD8a monospecific antibodies. huT cell purging is increased in the pancreas and liver of NRG-huPBL mice within 72 hours of receiving anti-huCD4/huCD8a bi specific antibody versus the combination of the monospecific anti-huCD4 and -huCD8a IgG4 (FIG. 37). 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. 37). 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 and 16).
DEFINING THE MECHANISMS OF PROTECTION OF CORECEPTOR THERAPY IN HUMANIZED MOUSE MODELS OF T CELL MEDIATED PATHOLOGY. Sphingosine-l -phosphate (S1P) is a major regulator of tissue egress by T cells. To test if coreceptor therapy-driven egress is mediated by S1P, the inventors employed FTY720. This drug blocks the function of the receptor for S1P; namely SlPrl . NRG-huPBL mice received the cocktail of 500 pg of monospecific anti-huCD4 and -huCD8a IgG4 plus/minus 10 mg/kg of FTY720. After 72 hours, the pancreas was harvested and T cell numbers determined via flow cytometry. As expected, T cells were markedly reduced in NRG- huPBL mice treated with anti-huCD4 and -huCD8a IgG4 versus control IgG4 (FIG. 39). In contrast, T cell-mediated purging of the pancreas by anti-huCD4 and -huCD8a IgG4 was blocked by administration of FTY720 (FIG. 39). These results demonstrate that coreceptor therapy-mediated tissue egress is regulated by S1P.
Notably, gene expression of SlPrl is regulated by the Foxol/KLF2 transcription pathway. To test if coreceptor therapy upregulates the Foxol/KLF2 transcription pathway
leading to enhanced SlPrl expression, NRG-huPBL mice were treated with the cocktail of monospecific anti-huCD4 and anti-huCD8a IgG4 or control IgG4. After 72 hours, CD4+ and CD8+ T cells were FACS-sorted from the pancreas. mRNA levels encoding KLF2, SlPrl and CCR7, another member of the transcription pathway were then measured by qRT-PCR. Relative to T cells from NRG-huPBL mice treated with control IgG4, a significant increase in the expression of genes encoding SlPrl, KLF2 and CCR7 was detected in both CD4+ and CD8+ T cells (FIG. 40). On the other hand, no increase in mRNA encoding CD3 was detected (FIG. 40), indicating that the effect of binding of anti-huCD4 and -huCD8a IgG4 is selective. Consistent with the gene expression results, surface expression of CCR7 and CD127, another member of the Foxol/KLF2 transcription pathway, was increased by anti-huCD4 and - huCD8a IgG4 as measured by flow cytometry (FIG. 38). Furthermore, surface expression of CD69 was markedly reduced (FIG. 38), indicating that coreceptor therapy suppresses T cell activation. Importantly, surface expression of CD127 was also increased on the surface of pancreatic CD4+ and CD8+ T cells in NRG-huPBL mice treated with the anti-huCD4 and anti- huCD8a bispecific antibody versus control IgG4 (FIG. 41). Together these findings indicate a scenario in which coreceptor therapy activates the Foxol/KLF2 transcription pathway leading to increased SlPrl expression, and enhanced responsiveness to S1P. This drives tissue egress of CD4+ and CD8+ T cells. This effect is seen with either the monospecific or bispecific anti- huCD4 and -huCD8a antibodies. Since S1P is a major regulator of T cell egress, the above mechanism of coreceptor therapy-induced purging is expected to be: 1) applicable to T cells targeting any tissue, and 2) therefore independent of the type of autoimmune disease or T cell- mediated pathology.
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 A-hydroxybenzotri azole (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 pl 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 (overnight 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 pl/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 {ref. Posthumus et al. (1990) J. Virol. 64:3304-3309)
LITERATURE REFERENCES. Timmerman etal. (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology. J. Mol. Recognit. 20:283-299 Langedijk etal. (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. 21). 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. 22) 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. 22 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. 21) and a heatmap (bottom panel FIG. 22). 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)
SPKLML SLKLENKE A (SEQ ID NO: 30)
PKLML SLKLENKEAK (SEQ ID NO: 31)
KLML SLKLENKEAK V (SEQ ID NO: 32)
LML SLKLENKEAK V S (SEQ ID NO: 33)
MLSLKLENKEAK V SK (SEQ ID NO: 34)
LSLKLENKEAKV SKR (SEQ ID NO: 35)
SLKLENKEAK V SKRE (SEQ ID NO: 36)
SET 2
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)
VELT2TASQKKSIQF (SEQ ID NO: 42)
LT2TASQKKSIQFHW (SEQ ID NO: 43)
2TASQKKSIQFHWKN (SEQ ID NO: 44)
IKNLKIED SDT YI2E (SEQ ID NO: 45)
NLKIED SDT YI2EVE (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 F SFPL AF TV A ALT GS (SEQ ID NO: 47)
(first 10) FPLIIKNLKAADSDT (SEQ ID NO: 48)
QGNFPLIIKAAKIED (SEQ ID NO: 49)
EV S VKRVTQ AAKLQM (SEQ ID NO: 50)
LML SLKLEN A AAK V S (SEQ ID NO: 51)
TGKLHQEVNAAVMRA (SEQ ID NO: 52)
SK S WITFDL A AKE V S (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 l5-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 CKK VVLGKKGDT VELT C (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)
CT2TASQKKSIQFHWKC (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 a-helical structure. Native Cys are replaced by Cys- acm (denoted“2”).
Sequences CSLWCQGNFPLIIKNLKIEDSD (SEQ ID NO: 67)
(first 10) CGNF CLIIKNLKIED SDT YI2E (SEQ ID NO: 68)
CP SKCNDRAD SRRSLWD QGNFP (SEQ ID NO: 69)
CQGNCPLIIKNLKIED SDTYI2 (SEQ ID NO: 70)
CKLNCRADSRRSLWDQGNFPLI (SEQ ID NO: 71)
CGPSCLNDRADSRRSLWDQGNF (SEQ ID NO: 72)
CWDQCNFPLIIKNLKIEDSDTY (SEQ ID NO: 73)
CLNDCADSRRSLWDQGNFPLII (SEQ ID NO: 74)
CADSCRSLWDQGNFPLIIKNLK (SEQ ID NO: 75)
CDQGCFPLIIKNLKIEDSDTYI (SEQ ID NO: 76)
SET 6
Mimic Type b-turn, mP2 CLIPS
Label BET
Description b-tum 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 b-tum formation. Cys are inserted on positions 1 and 22 and joined by means of mP2 CLIPS to stabilize the b-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)
CKGDTVELT2PGSQKKSIQFHC (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)
LTCTASQKKGGGIEDSDTYIC (SEQ ID NO: 90)
CTASQKKSIGGGIEDSDTYIC (SEQ ID NO: 91)
KGDTVELTCGGGDSDTYICEV (SEQ ID NO: 92)
DTVELTCT AGGGD SDT YICE V (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 pg/ml 25%SQ 25%SQ
CD4 mab 2 pg/ml 0. l%SQ l%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. 18.
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. 19). 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 b-strands. A schematic of this
approach is drawn in FIG. 20, 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. 23. 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. 24-26.
CONCLUSIONS. The monoclonal antibody CD4 mAh 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 mAh 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 A-hydroxybenzotri azole (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 pl 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 (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (SB A; Table 4) for one hour at 25°C. After washing, the peroxidase substrate 2,2’-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 pl/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 6. 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) ./. Virol. 64:3304-3309)
LITERATURE REFERENCES. Timmerman etal. (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology. ./. Mol. Recognit. 20:283-299 Langedijk etal. (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. 21). 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. 28 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. 28 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. 28) and a heatmap (bottom panel FIG. 28). 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 S QFRV SPLDRT WNLG (SEQ ID NO: 97)
(first 10) QFRV SPLDRTWNLGE (SEQ ID NO: 98)
FRV SPLDRTWNLGET (SEQ ID NO: 99)
RV SPLDRTWNLGET V (SEQ ID NO: 100)
V SPLDRTWNLGET VE (SEQ ID NO: 101)
SPLDRTWNLGET VEL (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 S QPL SLRPEGARP A A (SEQ ID NO: 117)
(first 10) L SLRPE ACR AGAGGA (SEQ ID NO: 118)
EGLDTQRFSAARLGD (SEQ ID NO: 119)
YFSHFVPVFAAAKPT (SEQ ID NO: 120)
TTPAPRPPTAGPTIA (SEQ ID NO: 121)
HFVPVFLPAAATTTP (SEQ ID NO: 122)
KPTTTPAPRAATPAP (SEQ ID NO: 123)
PTFLLYLSQAAPKAA (SEQ ID NO: 124)
S WLF QPRGAGGSPTF (SEQ ID NO: 125)
SDFRRENEGA AF C S A (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 l5-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 C SQFRV SPLDRTWNLGC (SEQ ID NO: 127)
(first 10) CFRV SPLDRT WNLGET C (SEQ ID NO: 128)
C V SPLDRTWNLGET VEC (SEQ ID NO: 129)
CPLDRTWNLGETVELKC (SEQ ID NO: 130)
CDRTWNLGETVELK2QC (SEQ ID NO: 131)
CTWNLGETVELK2QVLC (SEQ ID NO: 132)
CNLGET VELK2Q VLL S C (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) CLTLCDFRRENEGYYF 2 S AL SN (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 b-turn, mP2 CLIPS
Label BET.8
Description b-tum 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 b-tum formation. Cys are inserted on positions 1 and 22 and joined by means of mP2 CLIPS to stabilize the b-turn structure. Native Cys are replaced by Cys- acm (denoted“2”).
Sequences C SQFRV SPLDPGWNLGET VELC (SEQ ID NO: 147) (first 10) CQFRV SPLDRPGNLGET VELKC (SEQ ID NO: 148)
CFRV SPLDRTPGLGET VELK2C (SEQ ID NO: 149)
CRV SPLDRTWPGGETVELK2QC (SEQ ID NO: 150)
C V SPLDRTWNPGET VELK2Q V C (SEQ ID NO: 151)
C SPLDRTWNLPGT VELK2Q VLC (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 LGET VELKCGGGRENEGYYF C (SEQ ID NO: 157)
(first 10) ET VELKCQ V GGGRENEGY YF C (SEQ ID NO: 158)
VELKCQ VLLGGGRENEGYYF C (SEQ ID NO: 159)
LKCQ VLLSN GGGRENEGYYF C (SEQ ID NO: 160)
CQ VLL SNPT GGGRENEGY YF C (SEQ ID NO: 161)
LGET VELKC GGGNEGY YF C S A (SEQ ID NO: 162)
ET VELKCQ VGGGNEGYYF C S A (SEQ ID NO: 163)
VELKCQ VLLGGGNEGYYFCS A (SEQ ID NO: 164)
LKCQ VLLSN GGGNEGYYF C S A (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 7. Screening conditions
CD8 mab 1 pg/ml 25%SQ 25%SQ
CD8 mab 5 pg/ml l$SQ l%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. 18.
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. 19). 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 b-strands. A schematic of this approach is drawn in FIG. 20, 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. 29. 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. 30-31). In detail, antibody CD8 mAb systematically recognized linear and disulfide bridged peptides with core sequence 109 NEGYYFCSA 117 (SEQ ID NO: 168). Additionally, screening the antibody with looped peptides yielded an intensity profile with a single peak corresponding to peptides with core sequence 60 PRGAAASPTFLLY 72 (SEQ ID NO: 167).
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 (SEQ ID NO: 168) in three different peptide sets - linear, b-tum and disulfide bridges.
Binding to peptides with core sequence 60 PRGAAASPTFLLY 72 (SEQ ID NO: 167) was only established in peptide set containing single loop peptides lakj .pdb was used to assess structural features of identified epitopes (FIGS. 32-33). Even though 60 PRGAAASPTFLLY 72 (SEQ ID NO: 167) and 109 NEGYYFCSA 117 (SEQ ID NO: 168) 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
1. A method of treating a T cell mediated autoimmune/inflammatory disease in a subject comprising providing a bispecific antibody or antibody fragment having binding specificity for CD4 and CD8 to said subject, wherein said autoimmune/inflammatory disease is not diabetes.
2. The method of claim 1, wherein the subject is a human.
3. The method of claim 1, wherein the subject is a non-human mammal.
4. The method of claims 1-3, 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.
5. The method of claims 1-4, wherein the effects of providing are persistent.
6. The method of claims 1-5, wherein providing comprises administering said antibody or antibody fragment to said antibody.
7. The method of claims 1-5, wherein providing comprises genetic delivery of an RNA or DNA sequence or vector encoding the antibody or antibody fragment.
8. The method of claims 1-7, wherein providing results in reducing T effector cell number at a site of inflammation within the subject, such as CD4+ or CD8+ T cells.
9. The method of claim 8, wherein the site of inflammation is the subj ecf s liver, pancreas, salivary glands, ovaries, testes, skin, central nervous system, synovial tissue, gastrointestinal tract, thyroid, kidneys, lungs or eyes.
10. The method of claims 1-7, wherein providing results in an increase in Fox3+ Treg cell number and/or activity at a site of inflammation within the subject.
11. The method of claim 10, wherein the site of inflammation is the subj ecf s liver, pancreas, salivary glands, ovaries, testes, skin, central nervous system, synovial tissue, gastrointestinal tract, thyroid, kidneys, lungs or eyes.
12. The method of claims 1-11, further comprising administering to said subject a second therapy.
13. The method of claims 1-12, wherein said subject is selected from a neonate, a pediatric patient, a teenager, an adult or a patient over about 60 years of age.
14. The method of claims 1-13 wherein, said 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 (SEQ ID NO: 167).
15. The method of claims 1-13, 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.
16. The method of claims 1-13, or 15, 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.
17. The method of claims 1-13, 15 or 16, 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.
18. The method of claims 1-13, 15 or 16, 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.
19. The method of claims 1-13, 15 or 16, 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.
20. The method of claims 1-13, 15 or 16, 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.
21. The method of claims 1-13, 15 or 16, 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.
22. The method of claims 1-21, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment.
23. The method of claims 1-21, wherein said antibody is a chimeric antibody, a human antibody, an IgG antibody or a humanized antibody.
24. The method of claims 1-23, wherein said autoimmune/inflammatory disease is atopic dermatitis, autoimmune hepatitis, autoimmune retinopathy, celiac disease, chronic transplant rejection, Churg-Strauss syndrome, Graves disease, Graves ophthalmopathy, inflammatory bowel disease, graft-vv/.sv/.s-host disease, hashimoto's disease, juvenile idiopathic arthritis, multiple sclerosis, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, psoriasis, rheumatoid arthritis, Sarcoidosis, Sjogren's syndrome, Systemic Scleroderma, systemic lupus erythematosus, or ulcerative colitis.
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