Anticorpo Monoclonal

Introduction to Monoclonal Antibodies

Overview of the Lecture

• The speaker, Ronilson Brito, introduces himself and explains that he has been teaching immunology for over ten years. He started recording these videos for his undergraduate students during the pandemic in 2020.

• Ronilson expresses his desire to connect with viewers by showing his face on camera, emphasizing that the focus should remain on the content of the lecture.

Understanding Monoclonal Antibodies

• Monoclonal antibodies are a technology developed by two researchers in 1975. This technique is still widely used today for various diagnostic and research applications.

• Antibodies are produced by B lymphocytes (B cells). The speaker illustrates this process using a diagram of a plasma cell, which is an activated B cell that produces antibodies.

Activation and Function of Antibodies

Immune Response Mechanism

• Upon encountering microorganisms recognized as foreign antigens, B cells differentiate into plasma cells to mount an immune response.

• Plasma cells produce immunoglobulins (antibodies), which play a crucial role in opsonization—marking pathogens for destruction by other immune cells.

Structure of Antibodies

• The speaker discusses antibody structure, highlighting its composition from amino acids and differentiating between light chains (smaller) and heavy chains (larger).

• A visual representation shows two different colored regions: variable regions (green/yellow), responsible for binding to antigens, and constant regions (blue), which remain unchanged across different antibodies.

Variable Regions and Hypervariability

Detailed Structure Analysis

• The variable region contains segments known as hypervariable regions or complementarity-determining regions (CDRs). These segments are critical for antigen recognition.

Understanding Antigen Epitopes and Immune Response

The Role of CDR in Antibody Specificity

• The concept of antigenic epitopes is crucial for understanding the specificity of immunoglobulins, with CDR1, CDR2, and CDR3 playing significant roles in binding.

• Each Complementarity Determining Region (CDR) has a unique position; for instance, CDR1 does not bind where CDR2 does, emphasizing the precision of antibody-antigen interactions.

Polyclonal vs. Monoclonal Responses

• Immune responses are typically polyclonal; during infections, multiple B cells respond to various epitopes on an antigen.

• The initial immune response begins with innate immunity and transitions into adaptive immunity, specifically focusing on humoral responses involving antibodies.

Mechanism of B Cell Activation

• When encountering pathogens like Streptococcus pyogenes, specific B cells recognize distinct regions (epitopes), leading to their activation.

• Activated B cells undergo clonal expansion through mitosis, producing numerous identical cells that target the same epitope.

Diversity in Antibody Production

• Different B cells can recognize various epitopes on the same pathogen; this diversity ensures a robust immune response against multiple targets.

• Each activated B cell produces antibodies specific to its recognized epitope, highlighting the importance of specificity in immune responses.

Implications of Polyclonal Responses

• The presence of multiple epitopes means that no single B cell will suffice; instead, many are recruited to effectively combat an infection.

• This leads to a polyclonal response as each B cell generates antibodies tailored to different epitopes on the antigen.

Understanding Monoclonal Antibodies

Monoclonal Antibodies: Origins and Production

Introduction to Monoclonal Antibodies

• The discussion begins with the concept of monoclonal antibodies, which are produced in response to an antigen after injection into an animal. These antibodies target different epitopes of the same antigen.

Historical Context

• The origins of monoclonal antibody technology trace back to a pivotal publication in 1975 by scientists Georges Köhler and César Milstein, who later received the Nobel Prize for their groundbreaking work.

• Their discovery was significant enough to earn them the Nobel Prize in Medicine and Physiology, highlighting the importance of monoclonal antibodies in scientific research.

Mechanism of Monoclonal Antibody Production

• The production process starts with B cells, which are responsible for generating antibodies. Understanding how these cells function is crucial for developing monoclonal antibodies.

• B cells must be cultured under specific conditions that promote their survival and reproduction; however, they can be fragile and difficult to maintain in vitro.

Fusion Technique

• To enhance stability during culture, researchers fuse B cells with tumor cells. Tumor cells thrive better in culture due to their robust growth characteristics.

• This fusion results in hybridoma cells that combine properties from both parent cell types—B cells (antibody-producing) and tumor cells (long-lived).

Characteristics of Hybridomas

• Hybridomas are capable of producing large quantities of monoclonal antibodies because they inherit traits from both fused cell types.

• Following fusion, hybridomas initially contain two nuclei until they undergo mitosis, leading to segregation of chromosomes into daughter cells.

Genetic Considerations

• During cell division, there may be variations in chromosome distribution among daughter hybridoma cells. Some may lose essential genes required for antibody production.

• A small percentage of hybridomas may fail to rearrange immunoglobulin genes correctly, resulting in non-functional antibody production.

Monoclonal Antibody Production Process

Characteristics of B Cells and Tumor Cells

• Monoclonal antibodies possess characteristics of both B lymphocytes and tumor cells, leading to the concept of immortalized B cells due to their inherent properties.

Steps in Producing Monoclonal Antibodies

• The goal is to produce monoclonal antibodies specific to a known antigen (e.g., antigen X). This involves a series of experimental steps.

Step 1: Cloning B Cells

• The first step is obtaining clones of B cells responsible for antibody synthesis.

Step 2: Fusion with Tumor Cells

• The second step involves fusing these B cells with tumor cells, creating hybridoma cells that can produce antibodies.

Step 3: Selection of Hybridomas

• After fusion, it’s crucial to select the successfully fused hybridomas from those that did not fuse.

Step 4: Identifying Specific Clones

• Selected hybridomas are then screened for those producing the desired monoclonal antibody against antigen X.

Immunization Process in Mice

• To generate specific B cell clones, antigen X is injected into a suitable mammalian host (like a mouse), which will trigger an immune response.

Immune Response and Spleen Extraction

• Following immunization, after several weeks, the mouse undergoes euthanasia, and its spleen is harvested for B cells that have produced antibodies against antigen X.

Diversity of Antibody Specificity

• The spleen contains various B lymphocytes; while many will be specific to antigen X, others will target different antigens.

Final Steps in Hybridoma Selection

Understanding Cell Fusion in Tumor Research

The Basics of Cell Fusion

• Not all tumors are suitable for cell fusion; similar cells yield better results than different ones, which is why myeloma cells are chosen.

• Myeloma cells, derived from B-cells, are used because they have been successfully immortalized and can thrive in culture.

Characteristics of Myeloma Cells

• Myeloma cells lose their ability to produce antibodies but remain viable in culture, making them ideal for fusion with B-cells.

Steps in the Fusion Process

Step 1: Immunization and Collection

• After immunizing an animal to obtain B-cells, the next step involves fusing these with tumor (myeloma) cells.

Step 2: Co-Culturing Cells

• B-cells extracted from the spleen are placed in contact with tumor cells in a culture medium to encourage fusion.

Step 3: Inducing Cell Fusion

• Simple co-culturing does not induce fusion; additional stimuli are required to facilitate this process.

Techniques for Inducing Fusion

• Two main stimuli for inducing cell fusion include:

• A specialized medium enriched with polyethylene glycol (PEG), which increases membrane permeability.

• An electric pulse that also enhances membrane permeability, allowing the cells to fuse more easily.

Application of Electric Pulses

• Using an apparatus designed for electric pulses adjusts voltage and amperage to promote membrane fusion between B-cells and tumor cells.

Selection of Hybrid Cells

Identifying Successful Fusions

• After attempting cell fusion, it’s crucial to select only those hybridomas that successfully fused while eliminating non-fused B-cells and myeloma cells.

Observing Culture Outcomes

• In the culture, three types of cells may be present: unfused B-cells, unfused myeloma cells, and hybridomas. The goal is to isolate only the hybridomas.

Final Considerations on Antigen Response

• Following immunization against antigen X, there may be a higher likelihood of obtaining hybridomas that recognize this antigen due to prior stimulation during the process.

Understanding B Cell Culture and Hybridoma Technology

The Basics of B Cell Culture

• The initial step in culturing B cells involves separating them from the culture medium, as they do not thrive well without specific stimuli.

• B cells require mitotic stimulation (e.g., LPS) to survive; otherwise, they perish within a week or two even in nutrient-rich cultures.

• Tumor cells, unlike normal B cells, are immortal and can survive indefinitely in culture due to their inherent properties.

Selection of Hybridomas

• To isolate hybridomas from myeloma cells, a selective medium is used that only allows certain cells to thrive based on their metabolic capabilities.

• The selection medium contains components that must be metabolized by the surviving cells; this biochemical strategy is crucial for successful hybridoma cultivation.

Enzymatic Requirements for Survival

• A key enzyme required for survival in the selective medium is HGPRT (hypoxanthine guanine phosphoribosyltransferase), which is absent in myeloma cells.

• Myeloma cells cannot metabolize the selective medium due to the lack of HGPRT, leading to their death when exposed to it.

Fusion and Selection Process

• When B cells fuse with tumor cells to form hybridomas, they inherit characteristics from both parent cell types; importantly, B cells possess HGPRT.

• Only those hybridomas formed from the fusion of tumor and B lymphocytes can survive in the selective medium because they retain the necessary enzymatic function.

Final Steps in Hybridoma Isolation

• After allowing some time without stimulation, non-fused B cells will die off while hybridomas remain viable due to their unique properties.

• By replacing the culture with a selection medium that targets non-hybridoma populations, only desired hybridomas persist after treatment.

How to Isolate and Cultivate Cells?

Introduction to Cell Isolation

• The speaker discusses the challenge of finding specific cells within a mixed population, prompting a question on how to effectively isolate them.

• A method is introduced where cells are separated individually into a 96-well culture plate for identification.

Serial Dilutions for Single Cell Isolation

• Serial dilutions are performed to ensure that each well contains only one cell, which is crucial for accurate results.

• The process involves adding precise volumes of cell suspension into wells containing culture medium, ensuring proper dilution ratios.

Verification of Single Cell Presence

• To confirm the presence of a single cell in each well, an inverted microscope is used for visual inspection.

• If more than one cell is observed in a well, it must be discarded or separated to maintain the integrity of the experiment.

Cell Proliferation and Cloning

• Once isolated, individual cells begin to proliferate exponentially (1 becomes 2, then 4, etc.), forming clones known as hybridomas.

• All resulting cells from this process are genetically identical and produce monoclonal antibodies.

Antibody Production and Specificity Testing

• As these cloned cells continue to grow in nutrient-rich conditions, they also produce increasing amounts of antibodies over time.

• A critical question arises regarding whether all produced antibodies have the same specificity against antigen X; testing is necessary to confirm this.

Testing Antibody Specificity

Cultura Inclusiva e Placas de Elisa

Introdução às Placas de Elisa

• A apresentação inicia com uma descrição de uma placa de Elisa, destacando sua tampa que facilita o uso e a semelhança com placas de cultura.

Diferenças entre Placas

• É importante distinguir entre placas de Elisa e placas de cultura, pois cada uma tem um propósito específico no laboratório. A confusão pode levar a erros nos testes.

Sensibilização da Placa

• O processo envolve sensibilizar a placa de Elisa com antígenos específicos. Uma aula explicativa sobre a técnica de Elisa é mencionada como recurso adicional.

Adição dos Anticorpos

• Após sensibilizar a placa, anticorpos monoclonais são adicionados para verificar se eles reagem ao antígeno X. Este passo é crucial para identificar os anticorpos presentes.

Procedimento Experimental

• O procedimento inclui coletar sobrenadantes das culturas celulares onde os anticorpos são produzidos, garantindo que as células não morram durante o processo.

Identificação e Análise dos Anticorpos

Ligação dos Anticorpos aos Antígenos

• Quando um anticorpo monoclonal se liga ao antígeno X na placa, isso indica que o hibridoma correspondente está presente e deve ser cultivado mais.

Lavagem da Placa

• Durante a lavagem da placa, anticorpos não específicos são removidos. Apenas aqueles que se ligaram especificamente ao antígeno permanecem retidos na superfície da placa.

Visualização do Resultado

• Um segundo anticorpo conjugado com enzimas é adicionado para facilitar a visualização da reação. Isso permite identificar se houve ligação específica entre o anticorpo e o antígeno.

Conclusão do Processo Experimental

• O resultado final depende da presença ou ausência do anticorpo específico ligado ao antígeno X na placa. Se não houver ligação, o segundo anticorpo não terá lugar para se ligar.

Monoclonal Antibodies Production and Applications

Understanding Monoclonal Antibodies

• The specificity of the monoclonal antibody is crucial; it binds to a specific antigen, but issues arise if the secondary enzyme-linked antibody washes away during the process, leading to no positive results in ELISA tests.

• ELISA tests yield colorimetric results; lack of color indicates that the monoclonals derived from hybridomas are not specific enough for the target antigen, resulting in their loss during washing.

Analyzing ELISA Results

• The intensity of blue coloration in ELISA wells indicates stronger binding of monoclonal antibodies; selecting those with better adherence is essential for further analysis.

• After identifying successful wells, the next step involves expanding cultures containing hybridomas that produce effective monoclonal antibodies against a specific antigen.

Expanding Hybridoma Cultures

• To cultivate effective hybridomas, one must identify which ones produce desired antibodies and expand these populations in larger culture vessels.

• Successful wells (e.g., F10 and G8) from ELISA tests are expanded into larger cultures to increase antibody production.

Producing Monoclonal Antibodies

• Following expansion, large quantities of monoclonal antibodies can be harvested from cultured cells; some cells may also be preserved by freezing them in liquid nitrogen for future use.

• The labor-intensive process of producing monoclonal antibodies explains their high cost when purchased commercially for applications like flow cytometry.

Applications of Monoclonal Antibodies

• Monoclonal antibodies have diverse applications across laboratories including diagnostics, research, and therapeutic uses. Their development has been significantly influenced by pioneering work from German and English scientists.

Specific Uses in Research and Therapy

• They are utilized for cell phenotyping by marking specific cell types (e.g., CD4 T-cells), allowing identification through techniques like flow cytometry.

• In flow cytometry, conjugated fluorescent markers enable visualization of specific antigens on cell surfaces using monoclonal antibodies.

Therapeutic Applications

• Monoclonal antibodies play a role in treating various conditions such as cancer, inflammatory diseases, cardiovascular issues, autoimmune disorders, and transplant rejections.

Monoclonal Antibodies: Understanding Their Use and Challenges

Overview of Monoclonal Antibodies

• Monoclonal antibodies (mAbs) are specific antibodies produced from a single clone of cells, with 570 mAbs studied in clinical trials by commercial companies.

• Out of these, 79 therapeutic monoclonal antibodies have been approved by the FDA for various treatments, indicating significant progress in their application.

Therapeutic Applications and Side Effects

• The discussion raises concerns about the use of monoclonal antibodies in cancer treatment, particularly regarding potential side effects when administered to patients.

• Patients' immune systems may recognize murine (mouse-derived) antibodies as foreign, leading to an immune response against the treatment itself.

Immune Response and Consequences

• This immune response can result in adverse effects, including rapid elimination of the therapeutic antibody from the body due to pre-existing immunity.

• The formation of immune complexes can occur, potentially causing renal issues such as glomerulonephritis due to deposition in kidneys.

Strategies to Reduce Immunogenicity

• To mitigate immunogenicity, scientists developed differentiated monoclonal antibodies that resemble human proteins more closely.

• A graph illustrates that lower immunogenicity correlates with greater similarity to human tissues; murine antibodies are highly immunogenic compared to those modified for human compatibility.

Advances in Antibody Engineering

• Techniques like creating chimeric antibodies involve combining mouse and human components to reduce immunogenicity while retaining efficacy.

• Humanized antibodies further decrease immunogenic responses by increasing the proportion of human sequences within the antibody structure.

Humanized Antibodies and Their Nomenclature

Understanding Humanized Antibodies

• Humanized antibodies are derived from mouse models, specifically utilizing only the hypervariable regions known as Complementarity Determining Regions (CDRs), namely CDR1, CDR2, and CDR3.

• The remaining structure of the antibody is human-derived, resulting in a "humanized" antibody that exhibits lower immunogenicity compared to fully murine antibodies.

Immunogenicity Levels

• The discussion highlights varying levels of immunogenicity among different types of antibodies:

• Fully murine antibodies have 100% murine content.

• Chimeric antibodies contain about 30% murine components.

• Humanized antibodies consist of approximately 10% murine elements.

Nomenclature of Biopharmaceuticals

• The nomenclature for biopharmaceuticals involves a prefix indicating the drug's purpose followed by a suffix that denotes the source of the antibody. For example, "mab" signifies monoclonal antibody.

• The prefix consists of two syllables: one defining the bio-pharmaceutical name and another indicating its target disease.

Examples and Historical Context

• A table is referenced to illustrate how to interpret various names based on their prefixes and suffixes, helping identify their applications and origins (e.g., whether they are fully human or chimeric).

Milestones in Monoclonal Antibody Development

• The first therapeutic monoclonal antibody developed was muromonab in 1986 for acute transplant rejection; its use ended in July 2011.

• In 1994, the first chimeric antibody (OAB Siximab) was introduced for cardiovascular diseases.

• Notable developments include rituximab in 1997 for lymphoma treatment and daclizumab as a humanized option for transplant rejection prevention.

• The latest milestone mentioned is adalimumab (Humira), approved in 2002 for rheumatoid arthritis treatment.