Anti-MHC-peptide complex antibodies

Are you struggling to find a source of custom monoclonal antibodies that selectively bind MHC-peptide complexes? ProteoGenix has the solution. Our team of antibody experts can make your anti-MHC-peptide complex antibody using a range of animal species and services to fit your needs, including custom phage display libraries, single B cell clones, or hybridoma cell lines. Whatever your antibody needs, ProteoGenix will combine its resources, services, and 20 years of expertise to deliver reliable anti-MHC-peptide complex antibodies that consistently perform.

Antibody Phage Display

Antibody phage display involves collecting peripheral blood mononuclear cells (PBMCs) from immunized animal hosts (immune libraries) or unimmunized hosts (naïve libraries). Once PBMCs are collected, the antibody gene segments are converted into cDNA and cloned into a (bacteriophage equivalent of a plasmid) phagemid to make an antibody-phage fusion protein, exposing the antibody to the outer surface of the bacteriophage.

The target antigen is cloned and immobilized so it can bind the target antibody, a process called biopanning. Once bound, the DNA inside the bacteriophage (containing the antibody-related genes) is isolated, sequenced, and expressed to verify the antibody binds antigen with high affinity. Learn more about how using phage display library construction in less than 8 weeks.

Why Antibody Phage Display is Ideal for Making Human Therapeutic Antibodies

Antibody phage display is useful for generating monoclonal antibodies to target antigens that traditionally evade most antibody production methods. This includes cancer neoantigens, (such as MHC-peptide complexes), conserved antigens, and human antigens of critical importance. Therefore, if your biomedical research lab or pharmaceutical company is interested in fast-tracking human immunotherapy projects to treat cancer, autoimmunity, or other diseases, then antibody phage display technology is the appropriate method.

Phage Display Has No Antibody Species Limitations

Phage display technology has no species limitations. This means you can engineer custom phage display libraries in species ranging from humans to llamas. However, if you are interested in antibodies for clinical use, bypass the time-consuming process of antibody humanization by making your custom naïve library from human PBMCs. This convenience factor makes antibody phage display technology the preferred method to generate immunotherapeutic or diagnostic antibodies.

Phage Display Deliverables are Antibody DNA Sequences, Not Fragile Cell Lines

The other advantage antibody phage display offers over hybridoma and B-cell screening is the benefit of not having to keep fragile cell lines healthy and viable.

There are several ways labs can lose antibody-producing cell lines. The first is from Mycoplasma contamination. Mycoplasma is a slow-growing intracellular bacterium resistant to antibiotics found in cell medium. In fact, 15% to 35% of all cell lines tested are positive for Mycoplasma contamination. This type of biological contamination is notoriously difficult to treat, often destroying the entire cell stock.

Human error is another common reason laboratories lose cell lines. Stocks of cells must be stored long-term in liquid nitrogen. If frozen cell line stocks thaw because of a liquid nitrogen refill mistake then your precious stock of antibody-producing cells is gone.

Phage display overcomes these limitations because fragile cell clones are never produced. Instead, we identify three antibodies that bind your target anti-MHC-peptide complex and deliver the antibody sequences to you. The best part? You retain ownership of the antibody sequences. They are your intellectual property. Thus, you can modify the antibodies for therapeutic purposes or allow ProteoGenix to modify the antibodies for you.

ProteoGenix’s Pre-Built Human Phage Display Libraries

Convert your bench-side discovery into a life-saving bedside therapy using our pre-built human cancer or autoimmunity phage display libraries. ProteoGenix built the world’s first human cancer and human autoimmune phage display libraries to supercharge your immunotherapeutic projects, saving you time, money, and effort.

Our Pre-built Antibody Phage Display Library for use in Cancer Immunotherapy

The process of a healthy cell transforming into a cancer cell is associated with genetic instability that results in the expression of new antigens, termed neoantigens. These cancer neoantigens are presented by MHC-I causing T-cell activation and cancer cell death. However, when a cancer cell evades T-cell detection, uncontrolled cell division occurs triggering the formation if a malignant tumor.

Adaptive antibody responses are mounted against cancer neoantigens, including the cancer cell peptides presented by MHC I. The antibodies that selectively recognize MHC I-cancer peptide complexes are termed TCR-like antibodies.

TCR-like antibodies hold promising immunotherapeutic potential because they selectively bind cancer cells. Therefore, ProteoGenix captured the antibody repertoire from cancer patients in the form of an antibody phage display library to help scientists accelerate their therapeutic and diagnostic cancer projects (read more about TCR-like antibodies below). Therefore, if you discovered a novel MHC-antigen complex, ProteoGenix can generate your clinical monoclonal antibodies from start to finish using our pre-built human cancer library.

Our Pre-built Antibody Phage Display Library for use in Autoimmune Immunotherapy

One autoimmune disease risk factor is the expression of specific MHC II “risk” alleles by autoreactive antigen-presenting cells (autoAPCs). These MHC II alleles allow the escape of self-tolerance by inadvertently stimulating T-cells when presenting self-antigens.

Although rare, the adaptive immune system can recognize autoantigen presented by MHC II as foreign and build an antibody repertoire to target the MHC II-autoantigen complexes. As in cancer treatment, these TCR-like antibodies have the potential to selectively target and destroy harmful cells including autoreactive APCs.

Therefore, ProteoGenix developed the world’s first naïve human autoimmunity phage display library to help scientists accelerate the discovery of novel anti-MHC-autoantigen complexes for therapeutic and diagnostic applications. Once we identify monoclonal antibody clones that bind your MHC II-autoantigen complex, ProteoGenix can convert it into an antibody-drug complex antibody (ADC) or a bispecific antibody. Therefore, if you discovered a novel MHC-autoantigen complex, ProteoGenix can generate immunotherapeutic monoclonal antibodies from start to finish using our human autoimmune library.

Non-human Antibody Phage Display Libraries

ProteoGenix also offers non-human pre-built naïve antibody phage display libraries. If you are interested in making a custom anti-MHC-peptide complex antibody targeting a non-human MHC then choose from one of the following libraries:

• Rabbit naive antibody phage display library
• Camelid naïve antibody phage display library
• Dog naïve antibody phage display library.

Don’t see a prebuilt library compatible with your research?
Let us make your custom library. Custom libraries give you unlimited access to an array of antibodies that target different MHC-peptide epitopes. This service is perfect for those who know they will need a constant source of antibodies to target yet-to-be-identified MHC-peptide complexes in their disease model.

ProteoGenix Can Therapeutically Modify Your Phage Display Antibodies

We can adapt your monoclonal phage display antibody into a bispecific antibody or conjugate it to cytotoxic drugs (ADC antibody) to target diseased tissue. We can also adapt your custom anti-MHC-peptide complex antibody for diagnostic applications such as ELISA, flow cytometry, or clinical imaging.

Hybridoma Cell Line Generation

Generating a hybridoma cell line is a great way to make anti-MHC antibodies that bind MHC-peptide complexes with high affinity. The first step in producing a hybridoma cell line involves immunizing mice or rats with the purified target antigen. Next, we collect splenocytes from the immunized mice or rats and fuse the appropriate B-cells with a myeloma cell line.

We select the hybrid cells by screening the supernatant for antibodies that bind the target antigen by ELISA. Lastly, we subject the positive binders to limited dilutions to isolate individual cells and expanded them into colonies. We then screen each colony and further verify their potential to bind antigen by ELISA. The entire process takes 10 weeks from start to finish. Read more about ProteoGenix’s hybridoma technology

B-cell Screening

B-cell screening is a three-step process that produces high-affinity monoclonal antibodies. The first step is a 6- to 8-week process that starts with immunizing rodents with purified antigen assessed by SDS-PAGE. The immunization process has 4-6 rounds of injections using an optimized immunization protocol.

The second step is a 2-3 weeks process involving B-cell sorting and screening. This is achieved by isolating lymphocytes from the PBMC compartment and the spleen of immunized rodents. Lastly, the resulting B-cells are cultured in vitro and the supernatants are assessed for the presence of antibodies that bind the target antigen by ELISA. The resulting best positive antibodies are sequenced cloned, and expressed in XtenCHO cells. Antibody supernatants are further screened by ELISA to confirm the presence of high-quality monoclonal antibodies. Learn more about ProteoGenix’s B-cell screening and isolation methods.

The Challenges of Making an Anti-MHC Peptide Complex Antibody

Designing monoclonal antibodies that selectively bind MHC-peptide complexes, known as TCR-like antibodies, is a difficult task that few companies attempt. However, our antibody experts use their 25 years of average experience to design and troubleshoot the most daunting antibody engineering challenges to consistently deliver high-quality antibodies that exceed your expectations. With 30+ antibodies in preclinical and clinical trials and 3 therapeutic antibodies on the market, we know how to design high-performing antibodies that deliver exceptional results. Below are a few of the challenges associated with successfully designing anti-MHC-peptide complex antibodies.

MHC Genetic Diversity Among Species

MHC is a multiprotein complex consisting of four different gene products. There are thousands of different MHC alleles in vertebrates each being highly polymorphic. This means a vast array of genetic variability exists between MHC genes among the same species. This genetic variability makes it difficult to design an antibody that consistently targets MHC in the same species unless it is an inbred laboratory rodent strain such that each animal is homozygous for a particular MHC haplotype.

At ProteoGenix, we don’t just design antibodies that recognize MHC, we design antibodies that recognize MHC-peptide complexes. This adds another layer of complexity to the process because not only are the MHC genes highly diverse but so are the foreign and self-peptides they present to T cells.

Difficult to Reassemble a MHC-peptide Complex

Isolating the correct antibody from a phage display library requires large quantities of a purified antigen. Therefore, we must clone and modify the MHC genes so the protein products are soluble (not membrane-bound) and load the soluble MHC complex with chemically synthesized peptide epitopes. The soluble peptide-loaded MHC complex is incubated with antibody-expressing bacteriophages and immunoprecipitated from the solution. The unbound antibody-conjugated phages are washed away leaving behind clones that bind the target antigen. Each binding clone is validated for its capacity to bind antigen by ELISA, ensuring correct binding to the MHC-peptide complex and not empty MHC or MHC loaded with a non-specific peptide.

What is the Function of Major Histocompatibility Complex (MHC)?

Major Histocompatibility Complex (MHC) are protein complexes expressed on every nucleated cell in vertebrates. The protein products of MHC genes play a critical role in the adaptive immune response to pathogens and cancer by presenting peptide fragments to T-cells. This process, known as antigen presentation, is essential for stimulating T-cell immune responses during infection.

T cells bind the MHC loaded with a peptide epitope using their T cell receptor (TCR). T cell activation occurs when the TCR binds a peptide-loaded MHC ligand that it was not trained to identify during positive selection in the thymus. This recognition stimulates a signaling cascade within T cells, activating T cells and downstream effector functions.

MHC Genetic Diversity

The genes that encode major histocompatibility complex (MHC) are clustered in a large gene locus within vertebrate DNA. MHC alleles are polymorphic meaning the DNA sequences are genetically diverse between species of the same organism. In humans, the MHC locus contains 224 genes covering 3.6 megabase pairs on chromosome 6. While some genes in the locus are pseudogenes the other half have known immune functions.

Most mammals have MHC loci that contain a large amount of allelic diversity. The most diverse loci in humans are HLA-A, HLA-B, and HLA-C, each containing more than 5,000 genetically distinct DNA sequences. This diversity is observed at the DNA level and in the amino acid sequence of MHC-encoded genes.

Different Classes of MHC

There are three different classes of MHC genes expressed in vertebrates, MHC class I and MHC class II and the lesser well-known MHC III. MHC class I is expressed in most nucleated cells and anucleate cells such as platelets (but not red blood cells). This protein complex consists of a combination of four different polymorphic genes, whose protein products collectively form the functional complex known as MHC I. These genes encode proteins called an α1 and α2, that form the antigen binding domain, an α3 chain that anchors MHC I to the cell membrane, and a β macroglobulin chain.

MHC II is largely expressed in antigen-presenting cells (APC) such as macrophages, dendritic cells, and B cells. These APCs phagocytose microorganisms, process the proteins into smaller peptides in the phagolysosome, and load them onto MHC II. Vesicles containing MHC II/peptide complexes fuse with the outer membrane of the APC, allowing the peptide to be presented to the TCR of T cells. TCR stimulation promoted T cell activation and downstream effector functions.

The Use of Anti-MHC-Peptide Loaded Antibodies In Cancer Immunotherapy

T-cells surveil the body’s tissues to not only identify infected cells but also cancer cells. Cancer cell recognition occurs when the TCR of a CD8+ cytotoxic T cell recognizes the cancer cell peptide antigen presented by MHC I. This interaction stimulates the activation of the cytotoxic T cell resulting in the selective destruction of the cancer cell.

This mechanism plays a critical role in preventing malignant tumor formation. However, when this process fails, cancer cells can escape the MHC I TCR recognition axis resulting in rapid cancer cell proliferation, tumor formation, and tumor growth. Therefore, studying how cancer cells evade TCR detection has been an area of intense research focus over the years.

Antibodies for Cancer Immunotherapy

Scientists and physicians have leveraged advances in antibody technology to find exciting new ways to target and destroy cancer cells. TCR-like antibodies with and without a drug conjugate (antibody-drug conjugate) are specific immunotherapies that involve the use of custom-made antibodies that bind MHC I/cancer peptide complexes. The resulting interaction between the TCR-like antibody and the cancer cell peptide/MHC complex mimics that of a TCR by stimulating T cell proliferation, differentiation, cytokine, and chemokine secretion. Unlike TCRs, recognition of cancer cell peptide/MHC complexes by TCR-like antibodies can stimulate broader pharmacological mechanisms such as:

• Antibody-dependent cellular cytotoxicity (ADCC)
• Complement-dependent cytotoxicity (CDC)
• Antibody-dependent cellular phagocytosis (ADCP)
• Direct induction of apoptosis

In addition to these anti-tumor cell effects, TCR-like antibodies can also be adapted to mimic chimeric antigen receptors (CAR), allowing T cells to specifically recognize and target cancer cells containing the specific MHC/cancer peptide complex at the cell surface. Therefore, custom anti-MHC cancer peptide antibodies go beyond the scope of cancer diagnostics, they have exciting new immunotherapeutic potentials that ProteoGenix can help you leverage to advance your cancer therapy research needs.

The Use of Anti-MHC-Peptide Loaded Antibodies In Autoimmunity Immunotherapy

There are 80 different autoimmune diseases that have been characterized to date. These diseases are highly complex with many genetic, environmental, and immunological factors influencing the development and progression of disease. One autoimmune disease risk factor is the expression of specific MHC II alleles by autoreactive antigen-presenting cells (autoAPCs). These MHC II alleles, termed risk alleles, are thought to contribute to the escape of self-tolerance by inadvertently stimulating T-cells when presenting self-antigens, known in this context as autoantigens.

Antibodies for Autoimmunity Immunotherapy

Even though it is uncommon, the adaptive immune system has the ability to identify autoantigens presented by MHC II as foreign and develop a repertoire of antibodies to attack these complexes. TCR-like antibodies are antibodies that detect particular autoantigen MHC II-peptide complexes utilized in immunotherapies. These TCR-like antibodies have the ability to specifically target and eliminate dangerous cells, just like in cancer cell immunotherapies (i.e autoreactive APCs).

However, other therapeutic approaches are typically focused on restoring the balance between regulatory T cells and effector cells. This can be achieved by conjugating antibodies with immunosuppressive cytokines to induce autoAPC tolerance or creating a bispecific antibody that also binds inflammatory cytokines, thus reducing the abundance of these destructive cytokines at the site of pathology. Whatever your therapeutic goal ProteoGenix can help you achieve it using our first-of-its-kind autoimmune phage display library.