Humanized antibodies for therapy.
Protein engineering has made it possible to combine the binding sites of murine antibodies with human antibody regions. Antibodies constructed in this way have important advantages for therapy.
Animals↗
Biomedical subjects
C Queen
Publications and source records attributed to C Queen.
Anti-Tac-H, a humanized antibody to the interleukin 2 receptor, prolongs primate cardiac allograft survival.
High-affinity interleukin 2 receptors (IL-2Rs) are expressed by T cells activated in response to foreign histocompatibility antigens but not by normal resting T cells. To exploit this difference in IL-2R expression, anti-Tac-M, a murine monoclonal antibody specific for the IL-2R alpha chain, was used to inhibit organ allograft rejection. However, the use of murine anti-Tac as an immunosuppressive agent was limited by neutralization by human anti-murine antibodies and by weak recruitment of effector functions. To circumvent these difficulties, a humanized antibody to the IL-2R, anti-Tac-H, was prepared. This molecule is human with the exception of the hypervariable segments, which are retained from the mouse. In vivo survival of anti-Tac-H is 2.5-fold longer than simultaneously administered anti-Tac-M (terminal t1/2, 103 hr vs. 38 hr). In addition, anti-Tac-H is less immunogenic than anti-Tac-M when administered to cynomolgus monkeys undergoing heterotopic cardiac allografting. Specifically, all monkeys treated with anti-Tac-M developed measurable anti-anti-Tac-M levels by day 15 (mean onset, 11 days). In contrast, none of the animals receiving anti-Tac-H produced measurable antibodies to this monoclonal antibody before day 33. Finally, there was a prolongation of graft survival in the cynomolgus heterotopic cardiac allograft model in animals receiving anti-Tac. In animals that received anti-Tac-M, the allograft survival was prolonged compared to that of the control group (mean survival, 14 +/- 1.98 days compared to 9.2 +/- 0.48 days; P less than 0.025). Graft survival was further prolonged by anti-Tac-H with a mean survival of 20.0 +/- 0.55 days (compared to controls, P less than 0.001; compared to anti-Tac-M, P less than 0.02). There was no toxicity attributable to the administration of either form of anti-Tac. Thus, anti-Tac-H significantly prolonged allograft survival in primates, without toxic side effects, and may be of value as an adjunct to standard immunosuppressive therapy in humans.
Animals↗
Humanized antibodies for antiviral therapy.
Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.
Amino Acid Sequence↗
A recombinant, membrane-acting immunotoxin.
The anti-Tac antibody is known to bind to the p55 chain of the human interleukin 2 receptor. An immunotoxin was produced by genetically linking Clostridium perfringens phospholipase C (PLC) to the Fab domain of anti-Tac. For this purpose, the PLC gene, with its own promoter and signal sequence, was fused to the 5' end of the VHCH1 segment of the anti-Tac heavy chain gene. The anti-Tac light chain gene, with an attached bacterial signal sequence, was made part of the same transcriptional unit. Escherichia coli transformed with the construct secreted a recombinant immunotoxin, anti-Tac(Fab)-PLC, in an active form. Anti-Tac(Fab)-PLC bound to cells expressing the interleukin 2 receptor and inhibited protein synthesis, with a 50% inhibitory concentration of 0.02 nM (1.8 ng/ml).
Amino Acid Sequence↗
Anti-Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malignant and immune disorders.
The Mr 55,000 interleukin 2 receptor peptide (Tac; CD25) is not expressed by normal resting T-cells but is markedly up-regulated in adult T-cell leukemia and other malignancies, as well as on T-cells activated in normal immune, autoimmune, allograft, and graft-versus-host settings. Anti-Tac is a mouse monoclonal antibody directed against the Tac peptide. Our prior attempts to use this antibody in humans for antitumor therapy and immune regulation have been limited by weak recruitment of effector functions and neutralization by antibodies to mouse immunoglobulins. To circumvent these difficulties, we prepared several chimeric "humanized" anti-Tac antibodies by genetic engineering, including one "hyperchimeric" antibody (anti-Tac-II) in which the molecule is human except for the small hypervariable segments of the complementarity-determining regions retained from the mouse antibody. These constructs maintain high affinities for antigen and abilities to block T-cell activation and demonstrate new capabilities to perform antibody-dependent cell-mediated cytotoxicity, absent in the mouse anti-Tac. Hence, humanized antibodies have been developed to a tumor-associated antigen and activated T-cell marker with significant features that offer new therapeutic possibilities for select neoplastic and immune disorders.
Animals↗
An enhancer associated with the mouse immunoglobulin lambda 1 gene is specific for lambda light chain producing cells.
We have developed a system to study transcriptional regulation of the lambda immunoglobulin gene in a natural setting -- lambda light chain producing lymphoid cells. This assay system has allowed the detection of an enhancer element located 3' of the lambda gene coding sequence. The enhancer can stimulate transcription from the lambda promoter as well as from other immunoglobulin and unrelated promoters. Like all enhancers, the lambda enhancer can function in either orientation with respect to a promoter, but it is significantly more active in one orientation than in the other. The lambda enhancer is unusual in spanning at least 4000 bp of DNA sequence and containing several distinct subelements that have independent enhancer activity. The enhancer is also remarkable because it functions in lambda light chain producing cells but not in kappa chain producing cells. This fact can be interpreted to support a model of immunoglobulin gene rearrangement in which rearrangement follows and depends on transcriptional activation.
Animals↗
A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin.
Antibodies and growth factors have been chemically coupled to different toxins to produce cytotoxic molecules that selectively kill cells bearing appropriate antigens or receptors. Antibody-toxin conjugates (immunotoxins) produced using conventional chemical coupling techniques have several undesirable characteristics. The smallest binding unit of an antibody is an Fv fragment which consists of a light and heavy chain variable domain. Recently, active single chain Fv fragments of antibodies have been produced in Escherichia coli by attaching the light and heavy chain variable domains together with a peptide linker. Here we describe the construction and expression in E. coli of a single chain antibody toxin fusion protein, anti-Tac(Fv)-PE40, in which the variable regions of anti-Tac, a monoclonal antibody to the p55 subunit of the human interleukin-2 receptor, are joined in peptide linkage to PE40, a modified form of Pseudomonas exotoxin lacking its binding domain. Anti-Tac(Fv)-PE40 was very cytotoxic to two interleukin-2 receptor-bearing human cell lines but was not cytotoxic to receptor-negative cells.
Amino Acid Sequence↗
A humanized antibody that binds to the interleukin 2 receptor.
The anti-Tac monoclonal antibody is known to bind to the p55 chain of the human interleukin 2 receptor and to inhibit proliferation of T cells by blocking interleukin 2 binding. However, use of anti-Tac as an immunosuppressant drug would be impaired by the human immune response against this murine antibody. We have therefore constructed a "humanized" antibody by combining the complementarity-determining regions (CDRs) of the anti-Tac antibody with human framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac antibody sequence. In addition, a computer model of murine anti-Tac was used to identify several amino acids which, while outside the CDRs, are likely to interact with the CDRs or antigen. These mouse amino acids were also retained in the humanized antibody. The humanized anti-Tac antibody has an affinity for p55 of 3 x 10(9) M-1, about 1/3 that of murine anti-Tac.
Amino Acid Sequence↗
Transfection of an immunoglobulin kappa gene into mature human B lymphocytes.
We show in this report that the transcription induced by interleukin-2 or pokeweed mitogens of the kappa MOPC 41 immunoglobulin light-chain gene transfected into primary human or murine B lymphocytes initiates from a previously unobserved start site about 26 base pairs upstream of the start site used in myeloma cell lines.
Animals↗
Bacteriophage lambda N gene leader RNA. RNA processing and translational initiation signals.
The positions of RNA processing events mediated by RNase III and of two ribosome binding sites have been defined in an in vitro transcript of 321 nucleotides initiated at the major leftward promoter (PL) of bacteriophage lambda. Purified RNase III makes two specific endonucleolytic cleavages in the transcript at points 88 and 197 nucleotides from the PL start. The positions of these cuts suggest a secondary structure for the RNase III recognition site which is similar to other RNase III sites in which double-stranded cleavage occurs. Structure mapping experiments reveal a pattern of cleavages made in the PL transcript by nucleases specific for single- or double-stranded RNA that support the structure proposed for the RNase III stem and provide clear evidence for the stem-loop formed in the RNA at the position of the N recognition (nutL) site. Our finding that ribosomes bind efficiently in vitro to the region of the PL transcript which includes the AUG codon at position 223 supports other evidence that this triplet is the N protein start codon. The existence of an additional ribosome binding site in the N gene leader region just downstream from the nutL site identifies a second position possibly used for translational initiation or regulation. Its occurrence suggests potential roles for ribosome interaction and/or translation of the leader RNA in regulating phage development and N gene expression.
Bacteriophage lambda↗
Interferon-gamma induces light chain synthesis in interleukin 2 stimulated human B cells.
Human B cells were cultured without added lymphokines, with interleukin 2 (IL2) or interferon-gamma (IFN-gamma) alone, or with a combination of IL2 and IFN-gamma. Treatment with IL2 alone induced differentiation of the B cells, as shown by an increase in the number of immunoglobulin (Ig)-containing cells and plaque-forming cells, and by a larger amount of Ig secreted into the medium. In most of the induced Ig-containing cells, heavy chains but not light chains were detectable by cytoplasmic staining. Treatment with IFN-gamma alone did not stimulate B cell differentiation. However, a combination of IFN-gamma and IL2 was more effective than IL2 alone in inducing B cell differentiation, as measured by further increases in the number of plaque-forming cells and in total Ig secretion. Furthermore, after treatment with both IL2 and IFN-gamma, most cells that contained cytoplasmic heavy chains also contained cytoplasmic light chains. We conclude that IFN-gamma acts in synergy with IL2 in B cell differentiation by enhancing light chain synthesis, leading to secretion of Ig molecules.
Antibody Formation↗
Cell-type specific regulation of a kappa immunoglobulin gene by promoter and enhancer elements.
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Animals↗
A comprehensive sequence analysis program for the IBM personal computer.
We have developed a versatile program for the analysis of nucleic acid and protein sequences on the IBM Personal Computer. The program is interactive and self-instructing. It contains all the features generally found in sequence analysis programs on large computers, including extensive homology routines, as well as new procedures for the entry of sequence data. The program contains facilities to store and utilize the entire Nucleic Acid Sequence Data Bank. We have devised a new algorithm to find restriction enzyme sites, which allows our microcomputer program to find all sites on a small plasmid for 100 different enzymes in 1 to 2 minutes.
Amino Acid Sequence↗
Analysis of biological sequences on small computers.
We review a wide range of programs for micro- and minicomputers that facilitate the collection and analysis of DNA, RNA, or protein sequences. Special-purpose programs that perform a single function and general-purpose programs that perform a set of functions are both considered. The information presented should be useful to any biologist who wishes to obtain a computer program to aid in sequence investigations.
Amino Acid Sequence↗
Fine mapping of an immunoglobulin gene activator.
It has recently been shown that the promoter of a kappa immunoglobulin gene is activated for transcription by a downstream sequence element. Here we mapped the activating element to a resolution of about 20 base pairs by constructing a series of deletions in the cloned kappa gene. After transfection of each deleted gene into myeloma cells, a transient expression assay was used to measure the level of transcription from the kappa promoter. We found that the activating element extends through about 200 base pairs and encompasses a region of sequence that is conserved between mouse and human genes. As successively deeper deletions were made into the conserved region from either the 5' or 3' side, the activating ability was lost gradually rather than abruptly. Although several short segments in this region are homologous to sequences in viral enhancers, they did not seem to play a dominant role in the activating effect. We also found that the activating element remained functional when reversed in orientation or when moved upstream of the kappa gene.
Animals↗
Regulation of immunoglobulin transcription.
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Animals↗
Immunoglobulin gene transcription is activated by downstream sequence elements.
To examine the DNA sequences involved in regulation of kappa immunoglobulin gene expression, an assay was developed whereby transcription of a cloned, rearranged kappa gene could be detected following its transfection into antibody-secreting mouse myeloma cells. In a transient expression assay, RNA transcripts initiating 20 to 25 bp before the AUG translation initiator of the kappa gene were readily detected. The constant region (C kappa) and part of the J kappa-C kappa intron was then deleted from the rearranged gene and a downstream SV40 polyadenylylation signal was provided. No transcription was detected from the deleted gene upon transfection into myeloma cells, suggesting that after rearrangement the kappa variable region promoter is activated by sequences more than 2.6 kb downstream of J kappa. The rearranged kappa gene was also transfected into a nonlymphoid line of monkey cells and RNA transcripts were detected. Transcription in these cells, however, was at a relatively lower level than in myeloma cells, did not depend on the presence of downstream sequences, and initiated from a site about 10 bp downstream of the myeloma site. These experiments define a sequence downstream of the promoter that is necessary for accurate transcription of the kappa chain gene.
Animals↗
A vector that uses phage signals for efficient synthesis of proteins in Escherichia coli.
I have developed a plasmid vector pCQV2 for high-level expression of proteins in E. coli. The plasmid contains a very strong promoter and translation start point from bacteriophage lambda, and a restriction endonuclease site in which a gene may be placed under control of these initiation signals. The plasmid also contains the gene for a temperature-sensitive repressor of the lambda promoter, allowing 100-fold induction of protein expression. The vector pCQV2 has been used to synthesize SV40 small t antigen at the level of 5-10% of total E. coli protein, representing an order-of-magnitude improvement over previous methods.
Antigens, Viral↗