Monoclonal antibodies

Monoclonal antibodies

1. MONOCLONAL ANTIBODIES BY DR.AYUSH GARG PG JR1

2. Cancer Immunotherapy Immunotherapy is treatment that uses certain parts of a person’s immune system to fight diseases such as cancer. This can be done in a couple of ways : Stimulating your own immune system to work harder or smarter to attack cancer cells Giving you immune system components, such as man-made immune system proteins

3. Types of cancer immunotherapy The main types of immunotherapy now being used to treat cancer include: Monoclonal antibodies: These are man-made versions of immune system proteins. Antibodies can be very useful in treating cancer because they can be designed to attack a very specific part of a cancer cell. Immune checkpoint inhibitors: These drugs basically take the ‘brakes’ off the immune system, which helps it recognize and attack cancer cells .

4. Cancer vaccines: Vaccines are substances put into the body to start an immune response against certain diseases. We usually think of them as being given to healthy people to help prevent infections. But some vaccines can help prevent or treat cancer. Other, non-specific immunotherapies: These treatments boost the immune system in a general way, but this can still help the immune system attack cancer cells.

5. What are antibodies? An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies ( mAb ) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines.

6. Isotypes According to differences in their heavy chain constant domains, immunoglobulins are grouped into five classes, or isotypes : IgG , IgA , IgM , IgD , and IgE . IgG : IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid. IgA: IgA1 (90%) and IgA2 (10%), stomach and intestines IgM : normally pentamer , ocassionally hexamer , multiple immunoglobins linked with disulfide bonds IgD: 1% of proteins in the plasma membranes of B-lymphocytes, function unknown IgE : on the surface of plasma membrane of mast cells, play a role in immediate hypersensitive and denfensive for parasite

7. The structure of antibodies

8. History of Mab development 1890 Von Behring and kitasato discovered the serum of vaccinated persons contained certain substances, termed antibodies 1900 Ehrlich proposed the “ side-chain theory” 1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet expended. Almost the same time, Porter isolated fragment of antigen binding (Fab) and fragment crystalline (Fc) from rabbit y-globulin. 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine- aminopterin -thymidine (HAT) selection media. 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells 1990 Milstein produced the first monoclonal antibodies.

9. Monoclonal antibodies to treat cancer The immune system attacks foreign substances in the body is by making large numbers of antibodies . An antibody is a protein that sticks to a specific protein called an antigen . Antibodies circulate throughout the body until they find and attach to the antigen . Once attached, they can recruit other parts of the immune system to destroy the cells containing the antigen

10. VEGF PDGF HGF NRPs Eph TGF β bFGF Ang Endothelial cell & Pericyte BRAF MEK ERK RAS PI3K AKT mTor Tumor

11. Types of MoAbs Structure % Human Example Comments Mouse Tositumomab, Ibritumomab Radio-conjugates Chimeric 65 Cetuximab, Rituximab Humanized 95 Trastuzumab Human 100 Panitumumab Transgenic mice

12. The types of mAb designed Murin source mAbs : rodent mAbs with excellent affinities and specificities, generated using conventional hydrioma technology. Clinical efficacy compromised by HAMA(human anti murine antibody) response, which lead to allergic or immune complex herpersensitivities . Chimeric mAbs : chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource) Humanized mAbs : contained only the CDRs of the rodent variable region grafted onto human variable region framework

13. Nomenclature of MoAbs Last syllable is always – mab Next to last syllable -u- human (100%) : Panitum u mab - zu - humanized (95%) : Trastu zu mab -xi- chimeric (65%) : Ritu xi mab -o- mouse, -a- rat, -e- hamster, - i - primate : Tositum o mab Previous syllable - tu (m)- for tumor in general [ -ma(r)- breast, - pr (o)- prostate, -co(l)- colon, etc.] -ci(r)- for circulatory : Beva ci zumab

14. FDA-Approved “Naked” (Non-Conjugated) MoAbs Generic Name Brand Name Target Cancer(s) Alemtuzumab Campath CD52 CLL Bevacizumab Avastin VEGF Multiple Cetuximab Erbitux EGFR1 Colon, H&N Panitumumab Vectibix EGFR1 Colon Rituximab Rituxan CD20 Lymphomas Trastuzumab Herceptin HER-2 Breast

15. Types of monoclonal antibodies Naked monoclonal antibodies Naked mAbs are antibodies that work by themselves. There is no drug or radioactive material attached to them. These are the most common type of mAbs used to treat cancer . Most naked mAbs attach to antigens on cancer cells, but some work by binding to antigens on other, non-cancerous cells, or even free-floating proteins

16. Naked mAbs can work in different ways. Some boost a person’s immune response against cancer cells by attaching to them and acting as a marker for the body’s immune system to destroy them. An example is- Alemtuzumab which is used to treat some patients with chronic lymphocytic leukemia (CLL). Alemtuzumab binds to the CD52 antigen, which is found on cells called lymphocytes (which include the leukemia cells). Once attached,the antibody attracts immune cells to destroy these cells. Some naked mAbs boost the immune response by targeting immune system checkpoints.

17. Other naked mAbs work mainly by attaching to and blocking antigens on cancer cells (or other nearby cells) that help cancer cells grow or spread. For example , Trastuzumab is an antibody against the HER2 protein. Breast and stomach cancer cells sometimes have large amounts of this protein on their surface . When HER2 is activated, it helps these cells grow. Trastuzumab binds to these proteins and stops them from becoming active.

18. Conjugated monoclonal antibodies Monoclonal antibodies ( mAbs ) joined to a chemotherapy drug or to a radioactive particle are called conjugated monoclonal antibodies . The mAb is used as a homing device to take one of these substances directly to the cancer cells. The mAb circulates throughout the body until it can find and hook onto the target antigen. It then delivers the toxic substance where it is needed most. This lessens the damage to normal cells in other parts of the body . Conjugated mAbs are also sometimes referred to as tagged , labeled , or loaded antibodies.

19. Radiolabeled antibodies Radiolabeled antibodies have small radioactive particles attached to them . Ibritumomab tiuxetan is an example of a radiolabeled mAb . This is an antibody against the CD20 antigen, which is found on lymphocytes called B cells. The antibody delivers radioactivity directly to cancerous B cells and can be used to treat some types of non-Hodgkin lymphoma . Treatment with this type of antibody is sometimes known as r adioimmunotherapy (RIT).

20. Chemolabeled antibodies These mAbs have powerful chemotherapy (or other) drugs attached to them. They are also known as antibody-drug conjugates (ADCs). (The drug is often too powerful to be used on its own – it would cause too many side effects if not attached to an antibody.) Chemolabeled antibodies used to treat cancer include: Brentuximab vedotin an antibody that targets the CD30 antigen ( found on lymphocytes), attached to a chemo drug called MMAE . This drug is used to treat Hodgkin lymphoma and anaplastic large cell lymphoma. Ado- trastuzumab emtansine (also called TDM-1) , an antibody that targets the HER2 protein, attached to a chemo drug called DM1. It’s used to treat some breast cancer patients whose cancer cells have too much HER2.

21. A related drug known as denileukin diftitox is an immune system protein known as interleukin-2 (IL-2) attached to a toxin from the germ that causes diphtheria . Although it’s not an antibody, IL-2 normally attaches to certain cells in the body that contain the CD25 antigen, which makes it useful for delivering the toxin to these cells . Denileukin diftitox is used to treat lymphoma of the skin (also known as cutaneous T-cell lymphoma ). It’s also being studied for use against a number of other cancers.

22. Bispecific monoclonal antibodies These drugs are made up of parts of 2 different mAbs , meaning they can attach to 2 different proteins at the same time. An example is blinatumomab , which is used to treat some types of acute lymphocytic leukemia (ALL). One part of blinatumomab attaches to the CD19 protein, which is found on some leukemia and lymphoma cells. Another part attaches to CD3, a protein found on immune cells called T cells . By binding to both of these proteins, this drug brings the cancer cells and immune cells together, which is thought to cause the immune system to attack the cancer cells.

23. Common Side effects of mAb Fever Chills Weakness Headache Nausea Vomiting Diarrhea Low blood pressure Rashes

24. Some mAbs can have side effects that are related to the antigens they target. For example : Bevacizumab is an mAb that targets a protein called VEGF that affects tumor blood vessel growth. It can cause side effects such as high blood pressure, bleeding , poor wound healing, blood clots, and kidney damage . Cetuximab is an antibody that targets a cell protein called EGFR , which is found on normal skin cells (as well as some types of cancer cells). This drug can cause serious rashes in some people.

25. Immune checkpoint inhibitors to treat cancer An important part of the immune system is its ability to tell between normal cells in the body and those it sees as “foreign.” This lets the immune system attack the foreign cells while leaving the normal cells alone. To do this, it uses “checkpoints” – molecules on certain immune cells that need to be activated (or inactivated) to start an immune response. Cancer cells sometimes find ways to use these checkpoints to avoid being attacked by the immune system. But drugs that target these checkpoints hold a lot of promise as cancer treatments.

26. Treatments that target PD-1 or PD-L1 PD-1 is a checkpoint protein on immune cells called T cells. It normally acts as a type of “off switch” that helps keep the T cells from attacking other cells in the body. It does this when it attaches to PD-L1, a protein on some normal (and cancer) cells. When PD-1 binds to PD-L1, it basically tells the T cell to leave the other cell alone. Some cancer cells have large amounts of PD-L1, which helps them evade immune attack.

27. Monoclonal antibody treatments that target either PD-1 or PD-L1 can boost the immune response against cancer cells and have shown a great deal of promise in treating certain cancers. Examples of treatments that target PD-1 include: · Pembrolizumab ( Keytruda ®) · Nivolumab ( Opdivo ®) These have been shown to be helpful in treating melanoma of the skin, with some people having long-lasting responses to treatment. These treatments have also been shown to be helpful against non-small cell lung cancer in large studies. Smaller studies have shown promising early results against some other cancers as well, including kidney and colorectal cancer.

28. Treatments that target CTLA-4 CTLA-4 is another protein on some T cells that acts as a type of “off switch” to keep the immune system in check. Ipilimumab ( Yervoy ®) is a monoclonal antibody that attaches to CTLA-4 and stops it from working. This can boost the body’s immune response against cancer cells. This drug is used to treat melanoma of the skin. It is also being studied for use against other cancers. Because ipilimumab affects the immune system, it can sometimes cause serious or even life-threatening side effects. In fact, compared to drugs that target PD-1 or PD-L1, serious side effects seem to be more likely with ipilimumab .

29. Non-specific cancer immunotherapies and adjuvants Non-specific immunotherapies don’t target cancer cells specifically. They stimulate the immune system in a more general way, but this can still sometimes lead to a better immune response against cancer cells. Some non-specific immunotherapies are given by themselves as cancer treatments. Others are used as adjuvants (along with a main treatment) to boost the immune system to improve how well another type of immunotherapy (such as a vaccine) works. Some are used by themselves against some cancers and as adjuvants against others.

30. Cytokines Cytokines are chemicals made by some immune system cells. They are crucial in controlling the growth and activity of other immune system cells and blood cells Cytokines are injected, either under the skin, into a muscle, or into a vein. The most common ones are discussed here.

31. Interlukins Interleukins are a group of cytokines that act as chemical signals between white blood cells. Interleukin-2 (IL-2) helps immune system cells grow and divide more quickly. A manmade version of IL-2 is approved to treat advanced kidney cancer and metastatic melanoma. IL-2 can be used as a single drug treatment for these cancers, or it can be combined with chemotherapy or with other cytokines such as interferon- alfa . Using IL-2 with these treatments might help make them more effective against some cancers, but the side effects of the combined treatment are also increased.

32. Side effects of IL-2 can include flu-like symptoms such as chills, fever, fatigue, and confusion. Most people gain weight. Some have nausea, vomiting, or diarrhea . Many people develop low blood pressure, which can be treated with other medicines. Rare but potentially serious side effects include an abnormal heartbeat, chest pain, and other heart problems. Because of these possible side effects, if IL-2 is given in high doses, it must be done in a hospital. Other interleukins, such as IL-7, IL-12, and IL-21, are now being studied for use against cancer too, both as adjuvants and as stand-alone agents.

33. Interferons Interferons are chemicals that help the body resist virus infections and cancers. The types of interferon (IFN) are named after the first 3 letters of the Greek alphabet: · IFN- alfa · IFN-beta · IFN-gamma Only IFN- alfa is used to treat cancer. It boosts the ability of certain immune cells to attack cancer cells. It may also slow the growth of cancer cells directly, as well as the blood vessels that tumors need to grow.

34. IFN- alfa can be used to treat these cancers: · Hairy cell leukemia · Chronic myelogenous leukemia (CML) · Follicular non-Hodgkin lymphoma · Cutaneous (skin) T-cell lymphoma · Kidney cancer · Melanoma · Kaposi sarcoma

35. Side effects of interferons can include: · Flu-like symptoms (chills, fever, headache, fatigue, loss of appetite, nausea, vomiting) · Low white blood cell counts (which increase the risk of infection) · Skin rashes · Thinning hair

36. Alemtuzumab Indication Campath is indicated for the treatment of patients with chronic lymphocytic leukemia (CLL) who have been treated with alkylating agents and who have failed fludarabine therapy. Humanized monoclonal antibody directed against CD52 antigen Expressed on B & T lymphocytes Not expressed on bone marrow progenitor cells

37. 6 Mechanism of Action Lyses lymphocytes via Complement fixation Antibody dependent cell mediated cytotoxicity Induction of apoptosis Different mechanism of action from available cytotoxic chemotherapeutic agents CD52 Effector cell FC Receptor Campath Complement Leukemia Cell

38. Day 1 - 3 mg Campath 30 mg Campath 3x/week schedule e.g. Monday, Wednesday, Friday I.V. Administration Day 2 - 10 mg Campath Day 3 - 30 mg Campath Duration of Treatment 4-12 Weeks Based on Response

39. Trastuzumab Monoclonal antibody against epidermal growth factor receptor 2 (EGFR2, HER-2) Very effective against breast cancers in which HER-2 is “over-expressed” (more than usual amount per cell) (about 20% of all breast cancers) Often used in combination with chemotherapy

40. Trastuzumab : Mechanism of Action

41. 1. Trastuzumab mediates ADCC Once bound to the Fc domain of trastuzumab, the NK cells release substances… …that perforate the tumour cell membrane and promote cell death Nahta R, Esteva FJ. Breast Cancer Res 2006; 8: 215 Clynes RA, et al. Nat Med 2000; 6: 443-446 Gennari R, et al. Clin Cancer Res 2004; 10: 5650-5655 Arnould L, et al. Br J Cancer 2006; 94: 259-267

42. 2. Trastuzumab prevents formation of p95 HER2 Formation of the active p95 fragment, through proteolytic cleavage of the extracellular domain of HER2… …is prevented by trastuzumab Molina MA, et al. Cancer Res 2001; 61: 4744-4749 Nahta R, Esteva FJ. Cancer Lett 2006; 232: 123-138

43. 3. Trastuzumab blocks HER2-activated cell proliferation Trastuzumab interrupts this process HER2 signalling induces cell proliferation Nahta R, Esteva FJ. Cancer Lett 2006; 232: 123-138 Fry MJ. Breast Cancer Res 2001; 3: 304-312 Gershtein ES, et al. Clin Chim Acta 1999; 287: 59-67 Yakes FM, et al. Cancer Res 2002; 62: 4132-4141 Longva KE, et al. Int J Cancer 2005; 116: 359-367

44. 4. Trastuzumab inhibits HER2-regulated angiogenesis Trastuzumab inhibits this process HER2 signalling induces angiogenesis Izumi Y, et al. Nature 2002; 416: 279-280 Nahta R, Esteva FJ. Cancer Lett 2006; 232: 123-138 Wen XF, et al. Oncogene 2006; 25: 6986-6996 Klos KS, et al. Cancer 2003; 98: 1377-1385

45. Trastuzumab: 1 target 4 mechanisms of action Prevention of formation of p95 HER2 Inhibition of cell proliferation Activation of ADCC Inhibition of HER2-regulated angiogenesis

46. HER2 Herceptin Tumour cell + ADCC is a key mechanism of Herceptin’s antitumour activity in vivo ADCC FcgRIII NK cell Once bound to HER2, the Herceptin Fc domain recruits immune cells to target and destroy the tumour

47. Cetuximab Monoclonal antibody against epidermal growth factor receptor 1 (EGFR1) Effective in colon cancer and head and neck cancer; possibly useful in lung cancer Used with chemotherapy and with radiation therapy

48. IgG1 MAb ( chimerized ) Binds specifically to EGFR and its heterodimers Binds to EGFR with high affinity ( K d = 2.0 x 10 –10 M): 1 log higher than the natural ligand Following the recommended dose regimen (400 mg/m 2 initial dose/ 250 mg/m 2 weekly dose) , the mean half-life was 114 hours ( range 75-188 hours) Competitively inhibits ligand binding to EGFR Stimulates receptor internalization Blocks receptor dimerization, tyrosine kinase phosphorylation, and signal transduction

49. Bevacizumab Monoclonal antibody against vascular endothelial growth factor (VEGF), which stimulates angiogenesis (growth of new blood vessels into tumor ) Deprives tumors of the blood supply they need for growth and invasion Effective against cancers of colon, lung, breast, kidney, and brain Bevacizumab in combination with paclitaxel is indicated for the treatment of patients who have not received chemotherapy for their locally recurrent or metastatic HER2-negative breast cancer.

50. Rituximab: a mouse/ human chimeric anti- CD20 monoclonal antibody Murine variable regions bind specifically to CD20 on normal/ malignant B-cells Human K constant regions Human IgG1 Fc domain interacts with human effector mechanisms (ADCC, CDC) low immunogenicity

51. CD20 Expression in B-Cell Development Plasma cell Pluripotent stem cell Lymphoid stem cell Pre-B cell B cell Activated B cell Bone marrow Blood, lymph CD 20

52. Mechanism Of Action Malignant B-cell Complement CD20 CD20 Direct induction of apoptosis Killer Leukocyte

53. Side Effects Mild and transient, mainly during first infusion Fever, chills ( prevention) Temporary drop in blood pressure, dyspnea Rare: antibodies against rituximab

54. Monoclonal Antibodies In Development Epratuzumab ch806 Matuzumab CP-751,871 Nimotuzumab IMC-A12 Zalutumumab VEGF-Trap Pertuzumab IMC-18F1 Mapatumumab IMC-1121B Lexatumumab IMC-3G3 Volociximab Vitaxin Pemtumomab CNTO 95 Labetuzumab