Monoclonal Antibodies Can Be Constructed from IgGene Libraries

A quite different approach for generating monoclonal antibodies employs the polymerase chain reaction (PCR) to amplify the DNA that encodes antibody heavy-chain and light-chain Fab fragments from hybridoma cells or plasma cells. A promoter region and EcoRI restriction site (see Chapter 23) are added to the amplified sequences, and the resulting constructs are inserted into bacteriophage X, yielding separate heavy- and light-chain libraries. Cleavage with EcoRI and random joining of the heavy- and light-chain genes yield numerous novel heavy-light constructs (Figure 5-22).

This procedure generates an enormous diversity of antibody specificities—libraries with >1010 unique members have been obtained—and clones containing these random combinations of H + L chains can be rapidly screened for those secreting antibody to a particular antigen. The level of diversity is comparable to the human in vivo repertoire, and it is possible to demonstrate that specificities against a wide variety of antigens can be obtained from these libraries. Such a combinatorial library approach opens the possibility of obtaining specific antibodies without any need whatsoever for immunization.

However, the real challenge to bypassing in vivo immunization in the derivation of useful antibodies of high affinity lies in finding ways to mimic the biology of the humoral

CLI N ICAL FOCUS

CLI N ICAL FOCUS

Therapy for Non-Hodgkin's Lymphoma and Other Diseases by Genetically Engineered Antibodies

Lymphomas ae cancers of lymphatic tissue in which the tumor cells are of lymphocytic origin. There are two major forms of lymphoma: Hodgkin's lymphoma and non-Hodgkin's lymphoma. The less common form is Hodgkin's lymphoma, named for its discoverer, Thomas Hodgkin, an English physician. This unusually gifted early pathologist, who worked without the benefit of a microscope, recognized this condition in several patients and first described the anatomical features of the disease in 1832. Because many tissue specimens taken from patients Hodgkin suspected of harboring the disease were saved in the Gordon Museum of Guy's Hospital in London, it has been possible for later generations to judge the accuracy of his diagnoses. Hodgkin has fared well. Studies of these preserved tissues confirm that he was right in about 60% of the cases, a surprising achievement, considering the technology of the time. Actually, most lymphoma is non-Hodgkin's type and includes about 10 different types of disease. B-cell lymphomas are an important fraction of these.

For some years now, the major therapies directed against lymphomas have been radiation, chemotherapy, or a combination of both. While these therapies benefit large numbers of patients by increasing survival, relapses after treatment are common, and many treated patients experience debilitating side effects. The side effects are an expected consequence of these therapies, because the agents used kill or severely damage a broad spectrum of normal cells as well as tumor cells. One of the holy grails of cancer treatment is the discovery oftherapies that will affect only the tumor cells and completely spare normal cells. If particular types of cancer cells had antigens that were tumor specific, these antigens would be ideal targets for immune attack. Unfortunately, there are few such molecules known. However, a number of antigens are known that are restricted to the cell lineage in which the tumor originated and are expressed on the tumor cells.

Many cell-lineage-specific antigens have been identified for B lymphocytes and B lymphomas, including immunoglobulin, the hallmark of the B cell, and CD20, a membrane-bound phosphopro-tein. CD20 has emerged as an attractive candidate for antibody-mediated immunotherapy because it is present on B lymphomas, and antibody-mediated crosslinking does not cause it to down-regulate or internalize. Indeed, some years ago, mouse monoclonal antibodies were raised against CD20, and one of these has formed the basis for an anti-B-cell lymphoma immunotherapy. This approach appears ready to take its place as an adjunct or alternative to radiation and chemotherapy. The development of this anti-tumor antibody is an excellent case study of the combined application of immunological insights and molecular biology to engineer a novel therapeutic agent.

The original anti-CD20 antibody was a mouse monoclonal antibody with murine 7 heavy chains and k light chains. The DNA sequences of the light- and heavy-chain variable regions of this antibody were amplified by PCR. Then a chimeric gene was created by replacing the CDR gene sequences of a human 7I heavy chain with those from the murine heavy chain. In a similar maneuver, CDRs from the mouse k were ligated into a human k gene. The chimeric genes thus created were incorporated into vectors that permitted high levels of expression in mammalian cells. When an appropriate cell line was co-transfected with both of these constructs, it produced chimeric antibodies containing CDRs of mouse origin together with human variable-region frameworks and constant regions. After purification, the biological activity of the antibody was evaluated, first in vitro and then in a primate animal model.

The initial results were quite promising. The grafted human constant region supported effector functions such as the complement-mediated lysis or antibody-dependent cell-mediated cytotoxicity (ADCC) of human B lymphoid cells. Furthermore, weekly injections of the antibody into monkeys resulted in the rapid and sustained depletion of B cells from peripheral blood, lymph nodes, and even bone marrow. When the anti-CD20 antibody infusions were stopped, the differentiation of new B cells from progenitor populations allowed B-cell populations eventually to recover and approach normal levels. From these results, the hope grew that this immunologically active chimeric antibody could be used to clear entire B cell populations, including B lymphoma cells, from the body in a way that spared other cell populations. This led to the trial of the antibody in human patients.

The human trials enrolled patients with B-cell lymphoma who had a relapse after chemotherapy or radiation treatment. These trials addressed three important issues: efficacy, safety, and immunogenicity. While not all patients responded to treatment with anti-CD20, close to 50% exhibited full or partial remission. Thus, efficacy was demonstrated, because this level of response is comparable to the success rate with traditional approaches that employ highly cytotoxic drugs or radiation—it offers a truly alternative therapy. Side effects such as nausea, low blood pressure, and shortness of breath were seen in some patients (usually during or shortly after the initiation of therapy); these were, for the most part, not serious or life-threatening. Consequently, treatment with the chimeric anti-CD20 appears safe. Patients who received the antibody have been observed closely for the appearance of human anti-mouse-Ig antibodies (HAMA) and for human anti-chimeric antibody (HACA) responses. Such responses were not observed. Therefore, the antibody was not immunogenic. The absence of such responses demonstrate that antibodies can be genetically engineered to minimize, or even avoid, untoward immune reactions. Another reason for humanizing mouse antibodies arises from the very short half life (a few hours) of mouse IgG antibodies in humans compared with the three-week halflives of their human or humanized counterparts.

Antibody engineering has also contributed to the therapy of other malignancies such as breast cancer, which is diagnosed in more than 180,000 American women each year. A little more than a quarter of all breast cancer patients have cancers that over-express a growth factor receptor called HER2 (human epidermal growth factor receptor 2). Many tumors that over-express HER2 grow faster and pose a more serious threat than those with normal levels of this protein on their surface. A chimeric anti-HER2 monoclonal antibody in which all of the protein except the CDRs are of human origin was created by genetic engineering. Specifically, the DNA sequences for the heavy-chain and light-chain CDRs were taken from cloned mouse genes encoding an anti-HER2 monoclonal antibody. As in the anti-CD20 strategy described above, each of the mouse CDR gene segments were used to replace the corresponding human CDR gene segments in human genes encoding the human IgG1 heavy chain and the human k light chain. When this engineered antibody is used in combination with a chemotherapeutic drug, it is highly effective against metastatic breast cancer. The effects on patients who were given only a chemotherapeutic drug were compared with those for patients receiving both the chemotherapeutic drug and the engineered anti-HER2 antibody. The combination anti-HER2/chemotherapy treatment showed significantly reduced rates of tumor progression, a higher percentage of responding patients, and a higher one-year survival rate. Treatment with Herceptin, as this engineered monoclonal antibody is called, has become part of the standard repertoire of breast cancer therapies.

The development of engineered and conventional monoclonal antibodies is one of the most active areas in the pharmaceutical industry. The table provides a partial compilation of monoclonal antibodies that have received approval from the Food and Drug Administration (FDA) for use in the treatment of human disease. Many more are in various stages of development and testing.

Some monoclonal antibodies in clinical use

Monoclonal

antibody [mAB]

Nature of

Target

(Product Name)

antibody

(antibody specificity)

Treatment for

Muromonab-CD3

Mouse mAB

T cells

Acute rejection of liver, heart

(Orthoclone OKT3)

(CD3, a T cell antigen)

and kidney transplants

Abciximab

Human-mouse

Clotting receptor of platelets

Blood clotting during angioplasty

(ReoPro)

chimeric

(GP IIb/IIIa)

and other cardiac procedures

Daclizumab

Humanized mAB

Activated T cells

Acute rejection of

(Zenapax)

(IL-2 receptor alpha subunit)

kidney transplants

Inflixibmab

Human-mouse

Tumor necrosis factor, (TNF) a

Rheumatoid arthritis

(Remicade)

chimeric

mediator of inflammation. (TNF)

and Crohn's disease

Palivizumab

Humanized mAB

Respiratory Syncytial Virus (RSV)

RSV infection in

(Synagis)

(F protein, a component of RSV)

children, particularly infants

Gemtuzumab

Humanized mAB

Many cells of the myeloid lineage

Acute myeloid

(Mylotarg)

(CD33, an adhesion molecule)

leukemia (AML)

Alemtuzumab

Humanized mAB

Many types of leukocytes

B cell chronic

(Campath)

(CD52 a cell surface antigen)

lymphocytic leukemia

Trastuzumab

Humanized mAB

An epidermal growth factor

HER2 receptor-positive

(Herceptin)

receptor (HER2 receptor)

advanced breast cancers

Rituximab

Humanized mAB

B cells

Relapsed or refractory

(Rituxan)

(CD20 a B cell surface antigen)

non-Hodgkins lymphoma

Ibritumomab

Mouse mAB

B cells

Relapsed or refractory

(Zevalin)

(CD20, a B cell surface antigen)

non-Hodgkins lymphoma

SOURCE: Adapted from P. Carter. 2001. Improving the efficacy of antibody-based cancer therapies. Nature Reviews/Cancer 1:118.

SOURCE: Adapted from P. Carter. 2001. Improving the efficacy of antibody-based cancer therapies. Nature Reviews/Cancer 1:118.

Plasma cell #l

Plasma cell #l

VL-CL Vh-Ch1

VL-CL Vh-Ch1

Isolate mRNA's

Amplify by PCR

VT-CT

Insert into

Promoter

Not I f

Promoter

Vh-CH1

chain libraries Promoter EcoRl Not I

VT-CT

EcoRi

Prepare random Combinational libraries

Vh-CH1 Promoter

Heavy-light construct

Heavy-light construct

Not I

Vh-CH1 Promoter

FIGURE 5-22

Eco RI

Vt-CT

Not I

Not I

Vh-CH1 Promoter

EcoRI

Vt-CT

General procedure for producing gene libraries encoding Fab fragments. In this procedure, isolated mRNA that encodes heavy and light chains is amplified by the polymerase chain reaction (PCR) and cloned in \ vectors. Random combinations of heavy- and light-chain genes generate an enormous number of heavy-light constructs encoding Fab fragments with different anti-genic specificity. [Adapted from W. D. Huse et al., 1989, Science 246:1275.]

immune response. As we shall see in Chapter 11, the in vivo evolution of most humoral immune responses produces two desirable outcomes. One is class switching, in which a variety of antibody classes of the same specificity are produced. This is an important consideration because the class switching that occurs during an immune response produces antibodies that have the same specificity but different effector functions and hence, greater biological versatility. The other is the generation of antibodies of higher and higher affinity as the response progresses. A central goal of Ig-gene library approaches is the development of strategies to produce antibodies of appropriate affinity in vitro as readily as they are generated by an in vivo immune response. When the formidable technical obstacles to the achievement of these goals are overcome, combinatorial approaches based on phage libraries will allow the routine and widespread production of useful antibodies from any desired species without the limitations of immunization and hybridoma technology that currently complicate the production of monoclonal antibodies.

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