Therapeutic antibodies from the 19th to the 21st century GEOFF HALE Therapeutic Antibody Centre University of Oxford,

1. This lecture originally given as part of the FHS Pathology Course, Michaelmas Term Sir William Dunn School of Pathology, University of Oxford. � Geoff Hale, 2002, 2003. This lecture originally given as part of the FHS Pathology Course, Michaelmas Term Sir William Dunn School of Pathology, University of Oxford. � Geoff Hale, 2002, 2003.

2. Find all the slides, notes, bibliography etc at:users.path.ox.ac.uk/~seminars/halelibrary Contact me at: geoff.hale@path.ox.ac.uk

3. Antibodies have separate domains with different functions. Binding domains recognise the target antigen whereas the constant domains recruit various effector systems including complement and Fc receptors on killer cells. These systems are only activated by aggregation of multiple antibody molecules. Therefore soluble antibody can circulate in the body without effects, but when it binds to a target cell, the powerful effector systems are engaged.Antibodies have separate domains with different functions. Binding domains recognise the target antigen whereas the constant domains recruit various effector systems including complement and Fc receptors on killer cells. These systems are only activated by aggregation of multiple antibody molecules. Therefore soluble antibody can circulate in the body without effects, but when it binds to a target cell, the powerful effector systems are engaged.

4. Key properties of antibodies relevant to therapy Binding function Exquisite specificity High avidity from two binding sites Effector function Complement Fc receptors (ADCC) Long half life Liver FcRn receptor Carrier function for other drugs Besides their binding and effector functions, antibodies have another site which interacts with the FcRn (or �Brambell�) receptor. This rescues them from the normal process of metabolism and so endows antibodies with a much longer lifespan than other serum proteins.Besides their binding and effector functions, antibodies have another site which interacts with the FcRn (or �Brambell�) receptor. This rescues them from the normal process of metabolism and so endows antibodies with a much longer lifespan than other serum proteins.

5. Antibodies are different from conventional drugs Large size Unique biological activities Restricted tissue distribution Common structure Generic methodology Idiosyncrasies Potentially immunogenic Anti-idiotype therapy Anti-globulin responses There are advantages and disadvantages of each feature, but with careful design, the problems can be overcome and the unique advantages can be exploited.There are advantages and disadvantages of each feature, but with careful design, the problems can be overcome and the unique advantages can be exploited.

6. COMPTES RENDUSHEBDOMADAIRESDES SEANCESDE L�ACADEMIE DES SCIENCESTOME CENT-VINGTIEME1895 Traitment d�un cas de sarcome par la serotherapie J. HERICOURT et CH. RICHET This historic publication was the first to describe successful treatment of cancer using antisera. It was followed up with a second paper describing a series of similar patients. Tumour tissues were used to immunise rabbits or sheep and the antiserum was in turn administered to the patients.This historic publication was the first to describe successful treatment of cancer using antisera. It was followed up with a second paper describing a series of similar patients. Tumour tissues were used to immunise rabbits or sheep and the antiserum was in turn administered to the patients.

7. Conclusions of Hericourt and Richet Antibody therapy can ameliorate symptoms and give significant remission from disease. It is unlikely to cure advanced disease. In combination with other radical treatment, eg surgery, it may be a very effective adjuvant therapy for eliminating metastatic disease, possible leading to complete cure. One could draw exactly the same conclusions today about efforts to use monoclonal antibodies in cancer therapy!One could draw exactly the same conclusions today about efforts to use monoclonal antibodies in cancer therapy!

8. Modes of Antibody Therapy Passive Infectious diseases diphtheria, measles, rubella, hepatitis, tetanus, rabies Anti-venoms Active Cancer Cardiovascular disease Autoimmune disease and transplantation Anti-Rhesus One of the first really successful uses of antibody therapy was in the early 20th century when Paul Ehrlich developed and standardised anti-diphtheria serum. Although this is now superseded by vaccination, human immunoglobulin from immunised donors is still used to treat a number of infectious diseases and animal antisera are in wide use for the treatment of snake-bite or digoxin poisoning. It was harder to establish uses for polyclonal antibodies in the �active� category, but these diseases are receiving a lot of attention in the monoclonal era. A unique application of antibody therapy is the use of human anti-rhesus immunoglobulin for the treatment of Rh-negative mothers of Rh-positive babies to prevent sensitisation. This may work by rapid destruction of the foetal cells which enter the mothers circulation at the time of birth.One of the first really successful uses of antibody therapy was in the early 20th century when Paul Ehrlich developed and standardised anti-diphtheria serum. Although this is now superseded by vaccination, human immunoglobulin from immunised donors is still used to treat a number of infectious diseases and animal antisera are in wide use for the treatment of snake-bite or digoxin poisoning. It was harder to establish uses for polyclonal antibodies in the �active� category, but these diseases are receiving a lot of attention in the monoclonal era. A unique application of antibody therapy is the use of human anti-rhesus immunoglobulin for the treatment of Rh-negative mothers of Rh-positive babies to prevent sensitisation. This may work by rapid destruction of the foetal cells which enter the mothers circulation at the time of birth.

9. �The Pharmacy of the Future� A cartoon from the early 20th century with Behring, one of the early pioneers of antibody therapy supplying horse serum to his customers. Modern diagrams proposing antibody production in transgenic animals are not so different!�The Pharmacy of the Future� A cartoon from the early 20th century with Behring, one of the early pioneers of antibody therapy supplying horse serum to his customers. Modern diagrams proposing antibody production in transgenic animals are not so different!

10. Problems with polyclonal antisera Lack of Reproducibility Each batch could be different Complex Mixture Many different specificities Potential contaminants (eg viruses) Immunogenicity Animal proteins provoke immune response Loss of efficacy and serum sickness Despite many decades of effort, polyclonal antisera never became established for cancer therapy and a review in the 1960s concluded that nothing was likely to come from this line of research.Despite many decades of effort, polyclonal antisera never became established for cancer therapy and a review in the 1960s concluded that nothing was likely to come from this line of research.

11. The invention of monoclonal antibodies by Kohler and Milstein in 1975 revitalised interest in antibody therapy.The invention of monoclonal antibodies by Kohler and Milstein in 1975 revitalised interest in antibody therapy.

12. Pros and cons of monoclonal antibodies Unlimited supply So long as the cell line is stable Purity Single specificity = small doses Immunogenicity Mouse or rat = immunogenic Human monoclonals? Variable Potency Maybe less active than polyclonal Selection, cocktails and modifications The early enthusiasm for therapeutic monoclonal antibodies was soon tempered by the appreciation of new problems. Probably the most important issue is the selection of the most appropriate specificity and affinity.The early enthusiasm for therapeutic monoclonal antibodies was soon tempered by the appreciation of new problems. Probably the most important issue is the selection of the most appropriate specificity and affinity.

13. Antibodies for cell depletion (1) Natural mechanisms (naked antibody) Complement ADCC (macrophages, NK cells etc) Direct Induction of apoptosis Cytotoxic T cells The ability of antibodies to activate complement or ADCC depends on their isotype. In humans, the IgG1 isotype is generally the most potent. Some antibodies may bind to a receptor and mimic a natural ligand which delivers an apoptosis-inducing signal to the cell. There is probably an interplay between all three mechanisms because complement components and/or Fc receptors can contribute the cross-linking which is generally necessary for signal transduction. Rituximab (Rituxan�, anti-CD20) is a good example � this antibody is used for the treatment of lymphoma and there are various strands of experimental evidence showing the activation of complement, binding to Fc receptors and induction of apoptosis are all important for its mechanism of action. Cytotoxic T cells are not normally engaged by antibody, but special bispecific antibodies have been constructed which can cross-link T cells to target cells. These reagents can be extremely powerful because there are so many T cells available in the body. The ability of antibodies to activate complement or ADCC depends on their isotype. In humans, the IgG1 isotype is generally the most potent. Some antibodies may bind to a receptor and mimic a natural ligand which delivers an apoptosis-inducing signal to the cell. There is probably an interplay between all three mechanisms because complement components and/or Fc receptors can contribute the cross-linking which is generally necessary for signal transduction. Rituximab (Rituxan�, anti-CD20) is a good example � this antibody is used for the treatment of lymphoma and there are various strands of experimental evidence showing the activation of complement, binding to Fc receptors and induction of apoptosis are all important for its mechanism of action. Cytotoxic T cells are not normally engaged by antibody, but special bispecific antibodies have been constructed which can cross-link T cells to target cells. These reagents can be extremely powerful because there are so many T cells available in the body.

14. Antibodies for cell depletion (2) Artificial mechanisms (as carrier) Conventional drugs Immune activators Radioisotopes Toxins Enzyme/pro-drug (�ADEPT�) Viruses, genes etc A wide range of molecules have been attached to antibodies to enhance their ability to kill target cells. Some already approved for the market include (1) gemtuzumab ozogamycin (Myelotarg�), an anti-CD33 antibody coupled to the drug calcheamycin which is used for the treatment of acute myeloid leukaemia and (2) ibritumomab tiuxetan (Zevalin�), an anti-CD20 antibody coupled to Yttrium-90 which is used in the treatment of non-Hodgkin�s lymphoma.A wide range of molecules have been attached to antibodies to enhance their ability to kill target cells. Some already approved for the market include (1) gemtuzumab ozogamycin (Myelotarg�), an anti-CD33 antibody coupled to the drug calcheamycin which is used for the treatment of acute myeloid leukaemia and (2) ibritumomab tiuxetan (Zevalin�), an anti-CD20 antibody coupled to Yttrium-90 which is used in the treatment of non-Hodgkin�s lymphoma.

15. Genetic engineering of antibodies Reduce immunogenicity Chimeric, Humanised, Phage, Transgenic Modify effector function Aglycosyl, non-FcR binding Create fragments of complexes Hybrids, Bispecific Improve stability Human IgG4 Many approaches have been used to engineer antibodies to reduce immunogenicity. Therapeutic antibodies are generally chimeric or humanised but in future it is expected that so-called �fully human� antibodies made by phage display or in transgenic mice will be used. This area is complicated by a vast array of proprietary technology and competing patents. The constant regions of antibodies can also be modified to reduce or enhance effector function, to modify half-life and to create a wide range of fusion proteins with modified properties. Etanercept (Enbrel�) is a fusion protein consisting of an antibody Fc domain linked to the soluble domain of the human receptor for tumour necrosis factor (TNF). It is approved for the treatment of rheumatoid arthritis.Many approaches have been used to engineer antibodies to reduce immunogenicity. Therapeutic antibodies are generally chimeric or humanised but in future it is expected that so-called �fully human� antibodies made by phage display or in transgenic mice will be used. This area is complicated by a vast array of proprietary technology and competing patents. The constant regions of antibodies can also be modified to reduce or enhance effector function, to modify half-life and to create a wide range of fusion proteins with modified properties. Etanercept (Enbrel�) is a fusion protein consisting of an antibody Fc domain linked to the soluble domain of the human receptor for tumour necrosis factor (TNF). It is approved for the treatment of rheumatoid arthritis.

16. The first way to reduce immunogenicity was to link the variable domains from a mouse antibody with the constant domains of a human antibody. Chimeric antibodies on the market include rituximab (Rituxan�, anti-CD20), for treatment of lymphoma and infliximab (Remicade�, anti-TNF), for treatment of arthritis.The first way to reduce immunogenicity was to link the variable domains from a mouse antibody with the constant domains of a human antibody. Chimeric antibodies on the market include rituximab (Rituxan�, anti-CD20), for treatment of lymphoma and infliximab (Remicade�, anti-TNF), for treatment of arthritis.

17. A more subtle way to reduce immunogenicity was introduced by Greg Winter. Using site-directed mutagenesis, he introduced the hypervariable segments from a rodent antibody into a human Ig framework. This gives a humanised antibody with the same binding site as the original rodent antibody. Sometimes it is necessary to make a few changes to the framework in order to preserve the structure and binding affinity of the hypervariable loops. The first humanised antibody was alemtuzumab (Campath�, anti-CD52), now used for treatment of leukaemia.A more subtle way to reduce immunogenicity was introduced by Greg Winter. Using site-directed mutagenesis, he introduced the hypervariable segments from a rodent antibody into a human Ig framework. This gives a humanised antibody with the same binding site as the original rodent antibody. Sometimes it is necessary to make a few changes to the framework in order to preserve the structure and binding affinity of the hypervariable loops. The first humanised antibody was alemtuzumab (Campath�, anti-CD52), now used for treatment of leukaemia.

18. This diagram of a human IgG1 shows the hypervariable loops responsible for antigen binding which have been grafted from a rodent antibody.This diagram of a human IgG1 shows the hypervariable loops responsible for antigen binding which have been grafted from a rodent antibody.

19. Recombinant antibodies are made by inserting the desired segments of immunoglobulin genes into expression vectors. These are special plasmids which are constructed both to replicate in bacteria (to allow amplification of the DNA) and to express the Ig genes when transfected into mammalian host cells (to produce correctly assembled and fully glycosylated antibodies).Recombinant antibodies are made by inserting the desired segments of immunoglobulin genes into expression vectors. These are special plasmids which are constructed both to replicate in bacteria (to allow amplification of the DNA) and to express the Ig genes when transfected into mammalian host cells (to produce correctly assembled and fully glycosylated antibodies).

20. Filamentous bacteriophage provided the basis for a new method of antibody production. The wild type phage consists of DNA packed inside a protein coat. At one end there is another protein (pIII) which is required for binding and infection of bacteria. By incorporation of antibody gene elements into the pIII gene, phage display on their surface the antibody coded by the DNA inside, thus modelling the essential elements of a B lymphocyte. Filamentous bacteriophage provided the basis for a new method of antibody production. The wild type phage consists of DNA packed inside a protein coat. At one end there is another protein (pIII) which is required for binding and infection of bacteria. By incorporation of antibody gene elements into the pIII gene, phage display on their surface the antibody coded by the DNA inside, thus modelling the essential elements of a B lymphocyte.

21. From a large pool of lymphocyte DNA a library of variable region genes is isolated and inserted in random combinations into the phage DNA.From a large pool of lymphocyte DNA a library of variable region genes is isolated and inserted in random combinations into the phage DNA.

22. Following infection and amplification of the phage, a huge number of different specificities are expressed on the surface. The desired ones can be selected by binding to purified antigen and the resulting phage amplified again by replication in the bacterial host. After several cycles of such selection a population of antigen-specific phage is obtained, each containing the specific DNA which codes for the expressed Fv fragment. The DNA can then be isolated and used for antibody manufacture in bacterial or mammalian cells as required.Following infection and amplification of the phage, a huge number of different specificities are expressed on the surface. The desired ones can be selected by binding to purified antigen and the resulting phage amplified again by replication in the bacterial host. After several cycles of such selection a population of antigen-specific phage is obtained, each containing the specific DNA which codes for the expressed Fv fragment. The DNA can then be isolated and used for antibody manufacture in bacterial or mammalian cells as required.

23. Pros and cons of phage antibodies Start from human genes New specificities? Less immunogenic? Can screen a larger repertoire Loss of combinatorial specificities Bypass immunisation � naive library Low affinity Affinity maturation in vitro Extra steps, more immunogenic? Economical production in bacteria Only fragments Phage antibodies were promoted as faster to make and �fully human�, so not immunogenic. In practice there are still problems. The technology is not always so simple and a few companies (notably Cambridge Antibody Technology) hold key patents. Phage-produced antibodies still have unique idiotypes which are potentially immunogenic, especially if affinity matured in vitro. The first phage-produced antibody adalimumab (Humira�, anti-TNF) was approved at the end of 2002 for the treatment of rheumatoid arthritis. However, antiglobulin responses were observed in up to 12% of patients.Phage antibodies were promoted as faster to make and �fully human�, so not immunogenic. In practice there are still problems. The technology is not always so simple and a few companies (notably Cambridge Antibody Technology) hold key patents. Phage-produced antibodies still have unique idiotypes which are potentially immunogenic, especially if affinity matured in vitro. The first phage-produced antibody adalimumab (Humira�, anti-TNF) was approved at the end of 2002 for the treatment of rheumatoid arthritis. However, antiglobulin responses were observed in up to 12% of patients.

24. Antibodies from transgenic mice Companies Medarex, Affimetric, Translocus Disrupt mouse mu and kappa loci Ignore mouse lambda Introduce human mu and kappa Introduce most abundant human V genes 50% of repertoire from 7/87 genes Introduce human gamma constant regions as required IgG1 or IgG3 Can immunise, get affinity maturation and class switching A different way to make human antibodies which exploits the normal physiological selection systems is from transgenic mice. In a fantastic tour de force, companies such as Medarex and Affimetric have knocked out the majority of the mouse immunoglobulin genes and replaced them with human ones. Perhaps an important advantage of this system is the in vivo selection for stable combinations of heavy and light chains.A different way to make human antibodies which exploits the normal physiological selection systems is from transgenic mice. In a fantastic tour de force, companies such as Medarex and Affimetric have knocked out the majority of the mouse immunoglobulin genes and replaced them with human ones. Perhaps an important advantage of this system is the in vivo selection for stable combinations of heavy and light chains.

25. Source of antibodies on the market(according to a �market analyst�) Many reviewers portray mouse Ig domains as being �fully mouse� and human Ig domains as �fully human�. In fact of course there is considerable homology between species and considerable diversity within them so the table above is not really accurate.Many reviewers portray mouse Ig domains as being �fully mouse� and human Ig domains as �fully human�. In fact of course there is considerable homology between species and considerable diversity within them so the table above is not really accurate.

26. Antibody immunogenicity Immunogenic is not the same as antigenic Human antibodies are all different genetic variation (allotypes, V genes) somatic variation There is homology between species few C genes many V genes � can find a good match Because there are few constant region genes, there is a significant reduction in immunogenicity by makign chimeric antibodies. However, it is not necessarily the case that �humanised� or �fully human� antibodies offer a big improvement, because it is already possible to find V region genes in the human which are a close match to a particular mouse V region gene.Because there are few constant region genes, there is a significant reduction in immunogenicity by makign chimeric antibodies. However, it is not necessarily the case that �humanised� or �fully human� antibodies offer a big improvement, because it is already possible to find V region genes in the human which are a close match to a particular mouse V region gene.

27. Sequence homologies of variable regions www.path.cam.ac.uk/~mrc7 Examples of early humanised antibodies Humanised vs human myeloma 69-74% Murine vs human germline 68-75% Human myeloma vs human germline 78-91% Here are some examples of actual homology comparisons carried out by Mike Clark in Cambridge. Refer to his web site for further details. Note the difference between a human myeloma (somatically mutated) and the human germline.Here are some examples of actual homology comparisons carried out by Mike Clark in Cambridge. Refer to his web site for further details. Note the difference between a human myeloma (somatically mutated) and the human germline.

28. Conclusions from homology comparisons Some �chimeric� antibodies may be more homologous to germline than some �humanised� �Fully human antibodies may have drifted from germline sequence and be no more homologous than �chimeric� or �humanised� So all the efforts to humanise antibodies or make �fully human� antibodies might have been wasted!So all the efforts to humanise antibodies or make �fully human� antibodies might have been wasted!

29. Factors influencing the antiglobulin response Antibody structure Dose of antibody Number of injections Route of injection Immunocompetence of the recipient Cell binding or not? In reality, there are many factors which affect immunogenicity other than the structure of the antibody itself. Even the same antibody may provoke strong responses in one context (repeated sc administration) and not another (single dose iv). High doses can induce tolerance.In reality, there are many factors which affect immunogenicity other than the structure of the antibody itself. Even the same antibody may provoke strong responses in one context (repeated sc administration) and not another (single dose iv). High doses can induce tolerance.

30. Some observations Chimeric, humanised or �fully human� antibodies are less immunogenic than murine No published studies show difference in response between them Relative merits of the technologies depend on other technical or IPR factors Don�t believe all the hype from the companies who promote one technology over another!Don�t believe all the hype from the companies who promote one technology over another!

31. Engineering to reduce immunogenicity Disguise Cover up (eg PEG) Recruit a native Humanise Double agent Tolerise Suppose you were sending a spy into another country. There are several ways to avoid detection. Similarly there are different ways to engineer proteins to reduce immunogenicity. They may be �disguised� by covering antigenic epitopes with a relatively inert coat, eg polyethylene glycol. Native (ie human) proteins will be difficult to distinguish. But perhaps the most subtle approach is to allow them to engage with the host surveillance but in such a way that they are fully accepted, ie they behave like a �double agent�.Suppose you were sending a spy into another country. There are several ways to avoid detection. Similarly there are different ways to engineer proteins to reduce immunogenicity. They may be �disguised� by covering antigenic epitopes with a relatively inert coat, eg polyethylene glycol. Native (ie human) proteins will be difficult to distinguish. But perhaps the most subtle approach is to allow them to engage with the host surveillance but in such a way that they are fully accepted, ie they behave like a �double agent�.

32. Critical residues for effector function C1q binding Glu-318, Lys-320, Lys-322 FcR binding Leu-234, Leu-235, Gly-237 Glycosylation Asn-297 Mutation of these leads to loss of biological activity These are just a few of the residues identified to be involved in binding the molecules which are responsible for antibody effector functions. For a detailed analysis, refer to Shields et al, J. Biol. Chem. 276, 6591-6604 (2001).These are just a few of the residues identified to be involved in binding the molecules which are responsible for antibody effector functions. For a detailed analysis, refer to Shields et al, J. Biol. Chem. 276, 6591-6604 (2001).

33. Key residues for ligand binding are identified on this model of an IgG. Fc Receptors I, II and III as well as C1q all bind to a region in the hinge and CH2 domains. The presence of carbohydrate is essential to stabilise this structure and different glycoforms can modulate the binding affinity for these receptors. In contrast, the FcRn receptor which is responsible for the long half-life of IgG, binds to the CH2 domain at a site close to the Protein A binding site.Key residues for ligand binding are identified on this model of an IgG. Fc Receptors I, II and III as well as C1q all bind to a region in the hinge and CH2 domains. The presence of carbohydrate is essential to stabilise this structure and different glycoforms can modulate the binding affinity for these receptors. In contrast, the FcRn receptor which is responsible for the long half-life of IgG, binds to the CH2 domain at a site close to the Protein A binding site.

34. Increasing antibody affinity Advantages Increased potency = lower dose, fewer side effects and reduced cost? High affinity good for diagnostics Drawbacks Cross-reactivity increased = more side effects Poorer tissue penetration Potential for dose reduction limited by size of antigen sink It is sometimes suggested that high affinity antibodies are better for therapy, and so several companies have put effort into selection for the highest affinity. However, it is not always so desirable. Control and optimisation of antibody affinity may be a useful tool to permit the appropriate degree of tissue penetration.It is sometimes suggested that high affinity antibodies are better for therapy, and so several companies have put effort into selection for the highest affinity. However, it is not always so desirable. Control and optimisation of antibody affinity may be a useful tool to permit the appropriate degree of tissue penetration.

35. Monoclonals for tumour therapy Cell Depletion Rituxan, Campath (naked) Myelotarg (drug) Zevalin, Bexxar (radioisotope) Blocking receptors Herceptin Attacking vasculature Avastin, Erbitux Vaccination against idiotype Panorex? There are various ways of attacking tumours besides simply killing the target cells and the antibodies currently on the market exploit a range of different mechanisms. Possibly the most powerful is the provocation of a host immune response against the tumour, but this has proved to be very elusive.There are various ways of attacking tumours besides simply killing the target cells and the antibodies currently on the market exploit a range of different mechanisms. Possibly the most powerful is the provocation of a host immune response against the tumour, but this has proved to be very elusive.

36. Limiting factors Acute reactions Plasma antigen Antigenic modulation Immune response Tissue cross-reactivity Limited effector function Tumour localisation Tumour penetration There are many obstacles to antibody therapy for treating cancers, which is why so few antibodies have actually reached the market. Besides the scientific and technical difficulties listed above, the huge cost of clinical trials, the long time needed for evaluation and the complexity of patent issues are other barriers to be overcome.There are many obstacles to antibody therapy for treating cancers, which is why so few antibodies have actually reached the market. Besides the scientific and technical difficulties listed above, the huge cost of clinical trials, the long time needed for evaluation and the complexity of patent issues are other barriers to be overcome.

37. One of the most fundamental problems for large antibody molecules is the difficulty to access solid masses of cells, particularly when the blood supply is poor. Antibody tends to be bound by the first cells it encounters, especially if the affinity is high and the blood concentration is low. Cells deeper in the tumour can therefore escape attack.One of the most fundamental problems for large antibody molecules is the difficulty to access solid masses of cells, particularly when the blood supply is poor. Antibody tends to be bound by the first cells it encounters, especially if the affinity is high and the blood concentration is low. Cells deeper in the tumour can therefore escape attack.

38. Monoclonals approved for therapy (1) The major markets for therapeutic antibodies are in cancer and autoimmune disease. Some, such as Rituxan, Herceptin and Remicade already have annual sales close to the industry target of $1 billion and it is estimated that the global market for monoclonals will exceed $10 billion by 2008. Recent approvals, Avastin and Erbitux are targeted at the blood vessels supporting tumour growth and are used for treatment of metastatic colorectal cancer.The major markets for therapeutic antibodies are in cancer and autoimmune disease. Some, such as Rituxan, Herceptin and Remicade already have annual sales close to the industry target of $1 billion and it is estimated that the global market for monoclonals will exceed $10 billion by 2008. Recent approvals, Avastin and Erbitux are targeted at the blood vessels supporting tumour growth and are used for treatment of metastatic colorectal cancer.

39. Monoclonals approved for therapy (2) It is interesting to note that although there are 20 products in this list, there are only 15 different specificities. Few new targets have yet emerged from the genome sequencing projects of the 1990s.It is interesting to note that although there are 20 products in this list, there are only 15 different specificities. Few new targets have yet emerged from the genome sequencing projects of the 1990s.

40. CAMPATH-1 Monoclonal Antibodies CD52 antigen Expressed on: Approx 5% of lymphocyte surface Exceptionally lytic with human complement� Different isotypes and variants: Campath-1M rat IgM Campath-1G rat IgG2b Campath-1H human IgG1 Clinical applications: Leukemia and lymphoma Bone marrow and organ transplantation Autoimmune diseases Campath was developed by Herman Waldmann and his colleagues. It was the first humanised monoclonal and so it seems appropriate to use this an an example of the tortuous process of clinical development. The CD52 antigen is an unusual lipid-anchored glycoprotein with an exceptionally small protein component of only 12 amino acids. Campath is one of the most powerful antibodies for killing lymphocytes and has been tested in a wide range of clinical applications as an anti-tumour agent or an immunosuppressant.Campath was developed by Herman Waldmann and his colleagues. It was the first humanised monoclonal and so it seems appropriate to use this an an example of the tortuous process of clinical development. The CD52 antigen is an unusual lipid-anchored glycoprotein with an exceptionally small protein component of only 12 amino acids. Campath is one of the most powerful antibodies for killing lymphocytes and has been tested in a wide range of clinical applications as an anti-tumour agent or an immunosuppressant.

41. This is the CT scan of the first patient to be treated with the humanised version of Campath. She had a hugely enlarged spleen, with approx 4 kg of tumour cells, that mostly melted away within two weeks of treatment.This is the CT scan of the first patient to be treated with the humanised version of Campath. She had a hugely enlarged spleen, with approx 4 kg of tumour cells, that mostly melted away within two weeks of treatment.

42. The patient�s tumour was a B cell lymphoma. The tumour cells were characterised by a unique immunoglobulin gene rearrangement as shown on this blot of the restriction-digested DNA. A sample of bone marrow taken before treatment contained only tumour cells. In contrast, none of the tumour cell DNA could be detected in a sample taken shortly after Campath treatment.The patient�s tumour was a B cell lymphoma. The tumour cells were characterised by a unique immunoglobulin gene rearrangement as shown on this blot of the restriction-digested DNA. A sample of bone marrow taken before treatment contained only tumour cells. In contrast, none of the tumour cell DNA could be detected in a sample taken shortly after Campath treatment.

43. Story of Campath (1) 1980 CAMPATH-1M 1982 First bone marrow transplant 1985 Licensed to Wellcome 1986 CAMPATH-1G 1987 CAMPATH-1H Leukemia, transplantation, autoimmune disease 1994 Wellcome abandon CAMPATH-1M (rat IgM) was first developed for prevention of graft-versus-host disease by depletion of donor T cells from bone marrow used for allogeneic transplantation. It works by complement-dependent cell lysis. It was soon discovered that T-depleted bone marrow was easily rejected by the host and so Campath-1G was developed to purge the residual host T cells in vivo. It works by cell-mediated killing. The combination of antibodies, or even Campath-1G alone is still very effective for preventing the problems associated with stem cell transplants. However, their immunogenicity limited other uses and so in 1987 Campath-1H was made in a collaboration with Greg Winter. Wellcome (now part of Glaxo-Smith-Kline) carried out a lot of commercial development between 1985 and 1994, but abandoned the project when it seemed that Campath would not have blockbuster applications in lymphoma and arthritis.CAMPATH-1M (rat IgM) was first developed for prevention of graft-versus-host disease by depletion of donor T cells from bone marrow used for allogeneic transplantation. It works by complement-dependent cell lysis. It was soon discovered that T-depleted bone marrow was easily rejected by the host and so Campath-1G was developed to purge the residual host T cells in vivo. It works by cell-mediated killing. The combination of antibodies, or even Campath-1G alone is still very effective for preventing the problems associated with stem cell transplants. However, their immunogenicity limited other uses and so in 1987 Campath-1H was made in a collaboration with Greg Winter. Wellcome (now part of Glaxo-Smith-Kline) carried out a lot of commercial development between 1985 and 1994, but abandoned the project when it seemed that Campath would not have blockbuster applications in lymphoma and arthritis.

44. Story of Campath (2) 1994 Wellcome abandon BUT Investigators continue Multiple sclerosis etc 1997 Licensed to LeukoSite 2001 Campath approved for treatment of leukaemia In parallel with the commercial development, many academic investigators studied the uses of Campath for other indications including leukaemia, multiple sclerosis and vasculitis. The positive results from these studies encouraged LeukoSite, a small Biotech company, to take on the commercial license in 1997. LeukoSite was eventually sold to Millennium Pharmaceuticals who passed on the Campath license to ILEX Oncology. In 2001 Campath was approved for the treatment of chronic lymphocytic leukaemia. In parallel with the commercial development, many academic investigators studied the uses of Campath for other indications including leukaemia, multiple sclerosis and vasculitis. The positive results from these studies encouraged LeukoSite, a small Biotech company, to take on the commercial license in 1997. LeukoSite was eventually sold to Millennium Pharmaceuticals who passed on the Campath license to ILEX Oncology. In 2001 Campath was approved for the treatment of chronic lymphocytic leukaemia.

45. These magnetic resonance image (MRI) scans show an example of the lesions in the brain of a patient with multiple sclerosis (left). After a short course of Campath treatment very few new lesions are found for a period of at least 2 years (right).These magnetic resonance image (MRI) scans show an example of the lesions in the brain of a patient with multiple sclerosis (left). After a short course of Campath treatment very few new lesions are found for a period of at least 2 years (right).

46. Treatment of multiple sclerosis 27 patients 20 mg Campath for 5 days Disease modulation: Big reduction in brain lesions Very few new relapses Disability stabilised in half the patients Long-term immunity: Reduction in lymphocytes No significant infections 30% developed thyroid disease This slide summarises the key results from pilot studies of Campath by Alastair Compston and his team in Cambridge. A full-scale clinical trial is now planned by ILEX and their partners.This slide summarises the key results from pilot studies of Campath by Alastair Compston and his team in Cambridge. A full-scale clinical trial is now planned by ILEX and their partners.

47. This young lady was one of the first to be treated with Campath for vasculitis, an autoimmune disease which affects the blood vessels and causes a wide range of severe pathologic effects. Following her first course of treatment she made a dramatic recovery and went on to receive several other successful treatments with Campath when she later relapsed. However, she ultimately made a substantial anti-idiotype response and could not be further treated. Here she is pictured as guest of honour at the opening of the Therapeutic Antibody Centre in Oxford next to one of the fermentors used for manufacture of Campath and other antibodies.This young lady was one of the first to be treated with Campath for vasculitis, an autoimmune disease which affects the blood vessels and causes a wide range of severe pathologic effects. Following her first course of treatment she made a dramatic recovery and went on to receive several other successful treatments with Campath when she later relapsed. However, she ultimately made a substantial anti-idiotype response and could not be further treated. Here she is pictured as guest of honour at the opening of the Therapeutic Antibody Centre in Oxford next to one of the fermentors used for manufacture of Campath and other antibodies.

48. This lady also suffered from a form of vasculitis for which she was treated over several years with powerful immunosuppressive drugs including steroids and cyclophosphamide. By 1994 she had become refractory to these treatments which had also resulted in severe side-effects as exemplified by the photo taken in January. One of the major benefits of Campath, besides the improvement in her disease, was the ability to reduce or stop these other drugs. This lady also suffered from a form of vasculitis for which she was treated over several years with powerful immunosuppressive drugs including steroids and cyclophosphamide. By 1994 she had become refractory to these treatments which had also resulted in severe side-effects as exemplified by the photo taken in January. One of the major benefits of Campath, besides the improvement in her disease, was the ability to reduce or stop these other drugs.

49. Desirable properties for a target antigen Present on all tumour cells Abundantly expressed Not secreted Absent from normal tissues Complement and ADCC activation Extra features � immunotoxins, receptors Antigenic modulation limited The ideal monoclonal for cancer therapy would recognise a target with all these properties. Even then it still might not be a panacea! How many cell surface antigens meet these criteria? Scientists have been searching for suitable �tumour-specific� antigens for many years.The ideal monoclonal for cancer therapy would recognise a target with all these properties. Even then it still might not be a panacea! How many cell surface antigens meet these criteria? Scientists have been searching for suitable �tumour-specific� antigens for many years.

50. Potential for discovery of new targets Human genes 50,000 10% cell surface 5,000 10% tissue-specific 500 100 types of tissue Specific targets per tissue 5 10% have adequate density etc < 1 With some rough assumptions about the number of proteins coded by the human genome and the fraction which might be suitably expressed on the surface of a tumour, I estimate that there are very few truly tumour-specific antigens to be found if you limit the search to proteins. Perhaps there are more possibilities for carbohydrates since there are many possible glycoforms and these might well be modulated on the surface of tumour cells as a result of variations in activity of glycosyltransferases.With some rough assumptions about the number of proteins coded by the human genome and the fraction which might be suitably expressed on the surface of a tumour, I estimate that there are very few truly tumour-specific antigens to be found if you limit the search to proteins. Perhaps there are more possibilities for carbohydrates since there are many possible glycoforms and these might well be modulated on the surface of tumour cells as a result of variations in activity of glycosyltransferases.

51. Implications for development of new targets Majority of abundant tissue-specific antigens already discovered? Many Phase II/III are duplicates eg CD20, anti-TNF, CD18, CD3 Consider more widely distributed antigens? Isoforms CD45 Post-translational modifications Class 2 Relative abundance EMA Relative accessibility CD52 Many new clinical trials are still focused on similar or duplicate specificities to those already studied. To find new targets we may have to look again at antigens once thought to be too widely distributed.Many new clinical trials are still focused on similar or duplicate specificities to those already studied. To find new targets we may have to look again at antigens once thought to be too widely distributed.

52. Iterative clinical research The research of our group has gone in cycles from laboratory to clinic and back again, as antibodies are refined and developed to solve emerging problems. This was greatly facilitated in the past by a benign regulatory framework in the UK, but new European Directives will make this much more difficult in the future. The huge costs of regulatory compliance mean that government and charities will no longer be able to afford clinical trials of new drugs, and will be obliged to leave this to the pharmaceutical industry. We fear that this will limit development to only those compounds likely to have major markets; most likely palliative treatments for chronic conditions.The research of our group has gone in cycles from laboratory to clinic and back again, as antibodies are refined and developed to solve emerging problems. This was greatly facilitated in the past by a benign regulatory framework in the UK, but new European Directives will make this much more difficult in the future. The huge costs of regulatory compliance mean that government and charities will no longer be able to afford clinical trials of new drugs, and will be obliged to leave this to the pharmaceutical industry. We fear that this will limit development to only those compounds likely to have major markets; most likely palliative treatments for chronic conditions.

53. Find all the slides, notes, bibliography etc at: users.path.ox.ac.uk/~seminars/halelibrary Contact me at: geoff.hale@path.ox.ac.uk You are welcome to write to me if you have questions about therapeutic antibodies. I would be interested to receive feedback on the usefulness (or otherwise) of the references in my bibliography.You are welcome to write to me if you have questions about therapeutic antibodies. I would be interested to receive feedback on the usefulness (or otherwise) of the references in my bibliography.