OF BRMs IN INFECTIOUS DISEASES AND CANCER Monoclonal antibodies as immune modulators for cancer therapy

Monoclonal antibodies may modulate immune and/or biological responses 
alone, or as carriers of specific agents. Monoclonal antibodies directed against tumours may be indirectly 
cytotoxic by modulation of antibody-dependent, cell-mediated cytotoxicity or complement-mediated 
cytotoxicity. Monoclonal antibodies directed against certain tumour cell receptors may alter the biological 
behaviour of tumour cells such as blocking or downregulation of growth factors essential to tumour cell 
proliferation. Monoclonal antibodies directed to certain receptors on host immune cells. such as the CD3 
receptor on T lymphocytes. may activate those cells and increase their cytotoxicity. Antitumour monoclonal 
antibodies can serve as carriers of interferons, interleukin-2, tumour necrosis factor and other lymphokines 
and cytokines to modulate selectively the cytotoxic potential of immune cells in the vicinity of tumour cells. 
Cytotoxic chemotherapy agents conjugated to antitumour monoclonal antibodies may be processed 
differently so that they bypass certain mechanisms of drug resistance. The penultimate application of 
monoclonal antibodies in cancer therapy is to combine various monoclonal antibodies and immunoconjugates 
for selective combination therapy based on known antigenic tumour cell determinants and the 
status of the host immune system.


NAL USE 0 LY •DO NOT CO Monoclonal antibodies in cancer
T WO MAJOR LIMITATIONS TO EXISTING CANCER THERAPY are the lack of specificity of systemic modalities and the intra-and interpatient heterogeneity of cancer cells.Because of their natural specificity, antibodies against tumour-specific or associated antigens could be useful in overcoming these obstacles (l).In 1975, Kohler and Milstein (2) described the secretion of a monoclonal antibody by a B cell hybridoma.Subsequently, biotechnology companies have produced sufficient quantities of monoclonal antibodies for clinical investigation.Approaches for their use in cancer therapy are summarized below.

MONOCLONAL ANTIBODY ALONE
Indirect cytotoxicity: Some monoclonal antibodies n.x complement or interact with cytolytic and phagocytic cells to eliminate cells which are recognized by distinct surface molecules called antigens.Complement-dependent cytotoxicity (CDC) involves fiXation of complement Lo the Fe portion of the immunoglobulin (!g) molecule followed by activation of the complement cascade and the enzymatic puncturing of the tumour cell membrane resulting in cell death.Certain 'effector cells' such as monocytes, macrophages, granulocytes and certain lymphocytes have Fe receptors which bind to the Fe portion of Ig molecules.Cell killing via this combination is called antibody-dependent, cell-mediated cytotoxicity (ADCC).Monoclonal antibodies can bind to tumour cells and subsequently effector cells attach to the exposed Fe.Alternatively, injected monoclonal antibodies may attach to circulating effector cells and subsequently bind to tumour cells.Once such cells come in contact with tumour cells, they destroy tumour cell membranes enzymatically.

TABLE 1
For interaction with human complement, murine IgM is most efficient in CDC followed by the IgG3 subclass, while IgG2A.IgG1 and lgG2s murine antibodies are generally ineffective.For ADCC with murine monoclonal antibodies and human effector cells, the best results are obtained with murine subclasses IgG2A and IgG3.This correlates with the binding affinities of the Fe receptors for Ig molecules and the number of antigen-binding sites.For human antibodies and mouse-human chimeric antibodies, the best results in CDC are obtained with IgM > IgG1 > IgG3 > IgG2 > lgG4, while for ADCC this may be confined to human IgG1 and IgG3 monoclonal antibodies.Direct effects: Monoclonal antibodies may be directly cytotoxic, or impair tumour cell proliferation by inhibiting growth factors.They could also function indirectly with other components of the immune system as described above.
Tumour cells express increased numbers of receptors for molecules which augment their proliferation; many of these receptors are the products of oncogenes.Monoclonal antibodies directed against these receptors may compete with growth factors and/or downregulate receptors so that proliferative effects are lost.Much work in this area has focused on monoclonal antibodies directed against receptors for transferrin, interleukin-2 and epidermal growth factor.
B lymphocytes express the idiotype of their secretory Ig on their surface; idiotype is important in the activation and regulation of B cell proliferation.According to the 'network hypothesis', an anti-idiotype monoclonal antibody could inhibit proliferation of an aberrant B cell clone (3).This could be applied to therapy ofB lymphocyte malignancies.Immunization: Injection of murine monoclonal antibodies (ABI) into humans results in production of human antimouse antibodies (ABz) and, with repeated exposure, much of this is directed against the or hypervariable region of the mouse Ig.This human anti-idiotype antibody (ABz), in tum, induces an antiidiotype (AB3) response to the human antimouse antibody.Sometimes the AB3 is the mirror image of the murine monoclonal antibody which was originally injected.An antibody (ABz) can be raised against the idiotype of a murine antibody (ABJ) which reacts with tumour-associated antigen (4).A patient immunized with AB2 may produce an AB3 which reacts with the desired tumour antigen with the same reactivity as AB 1, except it would be a human antibody produced endogenously.This approach is similar to immunizing with tumour antigen except the idiotype may be more immunogenic.

CLINICAL TRIALS
Murine monoclonal antibodies have been used alone in hematopoietic malignancies (Table 1) and in patients with solid tumours (Table 2).In clinical trials with murine IgGz and IgG1 monoclonal antibodies, durable antitumour effects have seldom been seen, and complete responses have been exceedingly rare.With the exception of chronic lymphocytic leukemia and perhaps some B cell lymphoma patients, virtually all individuals exposed to murine monoclonal antibodies have developed an anlimouse immune response, typically within two weeks, which greatly alters antibody pharmacokinetics and the effects of repeated monoclonal antibody therapy.
Toxicities and side effects related to monoclonal antibody infusions have not been a significant problem (Table 3 cytopenia associated with fever, sweats and chills.In the absence of reactivity with circulating cells or circulating antigen, high doses of murine monoclonal antibodies have been given rapidly without side effects.However, cross-reactivity with normal tissues has resulted in antigen-specific toxicity. Immunoreactive antibody circulates for days to weeks depending on dose.human ant.imouse antibodies and tumour antigen burden.The degree of antibody concentration and binding in tumour increases over time and is dose-related.Circulating antigen does compete with tumour-bound antigen for antibody binding, but antigen in the circulation can be overcome by giving larger doses of monoclonal antibodies.However.immune complexes can form and are occasionally associated with significant toxicity.This has greatly limited the application of anti-idiotype monoclonal antibodies.Very large doses (grams) of monoclonal antibody may be needed in some situations.

USEO J.
Immunoconjugates: Radioisotopes , natural toxins , chemotherapy agents or other substances or cells can be chemically linked to monoclonal antibodies to fom1 'immunoconjugates'.There are several issues critical for any immunoconjugate.First is successful conjugation of as many cytotoxic molecules as possible (high specific activity) with retention of monoclonal antibody immunoreactivity and toxin cytotoxicity.Relative specificity for tumour is crucial.For certain toxins and drug conjugates, excellent in vitro specificity can be demonstrated despite many hours of exposure.Intracellular bi9availa bility is crucial for such conjugates because lhey must get into certain intracellular compartments for a cytotoxic effect.

Radiolabelled monoclonal antibodies:
The easiest immunoconjugates to make are radiolabelled antibodies which hold promise for radioimmunodetection and radioimmunotherapy (6).The ideal energy for gamma camera imaging efficiency is between 100 and 200 KeV.
The half-lives of some commonly used isotopes are 13 h for 123 iodine, 67 h for 111 indium.6 h for 99 technetium and eight days for 13 1 iodine.Technetium has superior imaging qualities, but its short half-life is a disadvantage for monoclonal antibodies which concentrate in tumour cells over time.Indium has good imaging qualities, but is difficult to conjugate chemically to monoclonal antibodies, is expensive, and in free form it concentrates in the reticuloendothelial system. 13\octine produces beta rays which could be useful for lherapy, but are not helpful for imaging. 123Iodine is not widely available, has a short half-life and is relatively expensive .Radiolabelled antibodies have detected tumour which is not detectable by standard radiological diagnostic techniques.There still is controversy regarding the advantages and disadvantages oft.he various alpha, beta and gamma emitters for radioimmunotherapy.Isotopes such as 188 rhenium, 90 yttrtum and 131 iodine release particles which penetrate several rnillimetres and, therefore, could be of value for antigen-negative cells.Alpha emitters such as 211 astatine and 212 bismuth are more powerful, but their energy dissipates over such a short range that killing would be limited to a single cell.Some radioimmunotherapy trials are summarized in Table 4. Therapy is hampered by prolonged circulation of radiolabelled monoclonal antibody which results in significant total body irradiation.Consequently, bone marrow suppression has been a major limitation to therapeutic intensity, while human antirnouse antibodies (I-lAMA) have limited repeated therapy.Immunotoxins: Potent toxins linked to antibodies are called immunotoxins.Toxicity is conveyed by whole toxins or subunits of natural substances such as ricin, abrin, modeccine, pokeweed antiviral protein, gelonin, saporin, diphtheria toxin and Pseudomonas exotoxin (7).Many of these consist of two protein chains, a B-chain which enables cell binding and an A-chain  which inhibits protein synthesis, generally at the level of the 60S ribosome.Most work has focused on the conjugation of the A-chain of ricin to monoclonal antibodies.In order to kill, these toxins must get inside cells.Such toxins are more cytotoxic than chemotherapy agents, but most toxins are much larger.For instance, ricin A-chain has a molecular weight of 32,000 Daltons.Also, natural toxins are antigenic themselves and induce a strong human immune response.
Clinical efforts with immunotoxins have been hampered by conjugation chemistry, large scale production issues and safety concerns.Results of some published trials are summarized in Table 5. HAMA, as well as antibodies against ricin A-chain, and a dose-limiting toxicity of a capillary leak syndrome martifested by normal liver function with hypoalbuminemia and edema have limited therapy.Some investigators have   Chemoimmunoconjugates: If sufficient chemotherapy could be delivered by monoclonal antibodies to tumour cells, greatly decreased toxicity and improved efficacy of chemotherapy would result.Cellular intemalization of a drug attached to a monoclonal antibody decreases resistance to the agent, possibly by decreasing efflux of free drug (8).Through the years, investigators have explored conjugation of various agents.Many efforts were limited by inefficient yields, drug cross-linking, impaired immunoreactivity and limited specific cytotoxicity.More recently, attention has been directed to the use of linkers such as cisaconitate, glutaraldehyde, albumin and short amino acid sequences.These approaches are limited by the number of free amino groups.particularly on lysine residues.Sitespecific linkage to carbohydrate groups on the Fe portion of the Ig molecules may allow for attachment of additional drug for some antibodies.Some linker approaches neutralize the activity of the drug while it is linked, but activity is restored after acid hydrolysis inside the cell or in the relatively acidic environment around hypoxic tumour cells.Such conjugates can exhibit specific cytotoxicity and are as effective or more effective than equivalent amounts of free drug in vitro and are superior to drug alone or drug plus antibody in animal tumour models (8).There is only limited clinical experience with drug-immunoconjugates (Table 6).Chemistry and large scale production are persistent problems.
Cellular conjugates: Heleroconjugates or bifunctional monoclonal antibodies may be used to direct activated T cells to tumours.Monoclonal antibodies directed against the T cell receptors, CD3 or CD2, activate those cells into a cytotoxic state so that they will kill if they come in close contact with tumour cells, especially in the presence of interleukin-2.Anti-CD2 or anti-CD3 monoclonal antibodies can be chemically linked to monoclonal antibodies which react with tumour antigen to produce a heteroconjugate.Bifunctional monoclonal antibodies in which one arm of the molecule is bound to CD2 or CD3 and the other to a tumour-associated antigen can also be used.These approaches are promis• ing in vitro.

MONOCLONAL ANTIBODIES WITH OTHER BIOLOGICAL RESPONSE MODIFIERS
There is good rationale for using monoclonal antibodies in combination with other biological response modifiers .For instance, gamma-interferon increases the expression of Fe receptors on various effector cells and thus enhances ADCC.Interleukin-2 and tumour necrosis factor enhance cell-mediated cytotoxicity.Various interferons increase the expression of certain tumour-associated antigens.Vasoactive agents , such as interleukin -2, tumour necrosis factor or hyperthermia, may have vascular effects which allow more molecules to penetrate the tumour.Only a few pilot sludies have been published (Table 7).

ISSUES AND FUTURE DIRECTIONS
The human anti-lg response is a major limitation for repeated therapy with murine monoclonal antibodies and appears to be an important consideration in the use of human and mouse-human chimeric monoclonal antibodies (9) .An increasing number of clinical trials are being conducted 'humanized' or human monoclonal antibodies.Depending upon the specific clinical goal, there are rationales for the use of antibody fragments rather than whole antibody.Similarly, there are some situations in which a heterofunctional antibody may be preferable to a monospecific bivalent antibody.Tumour cell heterogeneity is a major problem which may be overcome by using combinations of antibody.Monoclonal antibody immunoconjugates may offer the CAN J INFECT DIS V OL 3 SUPPL B AUG UST 1992 0 J•DO co Monoclonal antibodies in cancer greatest opportunity to use combined modality therapy to overcome tumour heterogeneity (Figure 1).

Figure 1 )
Figure 1) Potential combination monoclonal antibody therapy for cancer.Various antigens are depicted on the tumour cell surface representing the heterogeneity q[ cancer.Bifunctional antibodies are depicted with one Fab directed to a tumour antigen and the other to a biological response modifier (IFN Interferon.TNF Tumour necrosis factor.IL-2 lnterleukin-2).One antibody has an Fab directed to a CD2 or CD3 receptor on T cells which results in their activation and enhanced cytotoxicity.The Fe portions q{the antibodies are attached to a variety of cytotoxic substances including complement, effector cells {granulocytes, monocytes, killer T cells) of the immune system and cytotoxic agents such as toxins, radioisotopes and chemotherapy agents {DNR Daunorubicin) {Reproduced with pem1issionfrom reference 1)

antibody lmmunogJobulin Disease Author Response rate
ALL Acute lymphocytic leukemia: AML Acute myelogenous leukemia: Ch Chimeric: CLL Chronic lymphocytic leukemia: CTCL Cutaneous T cell lymphoma: Mu Murine DILLMAN ONLY

TABLE 2 Published trials of monoclonal antibody passive therapy in solid tumours
Ch Chimeric: Hu Human: Mu Murine

TABLE 3 Summary of side effects and toxicities associated with murine monocolonal antibody infusions* Percentage of Percentage of Toxicity
(5)r 291 infusions in 177 patients.20difference malignancies.19murine and three human monoclonal antibodies(5)

TABLE 4
'Fractions represent responses (complete and partial) over number of patients treated.CLL Chronic lymphocytic leukemia: CTCL Cutaneous T eel/lymphoma; EGFR Epidermal growth factor; HAMA Human antimouse antibodies: HMFG Human milkfat globulin: IP Intraperitoneal; PR Partial response; RT Radiotherapy