Immunotoxins to the HER-2 oncogene product: Functional and ultrastructural analysis of their cytotoxic activity

Two immunotoxins were prepared using monoclonal antibodies (mAb) directed towards two distinct epitopes of the gp185^HER-2 extracellular domain, and the type I ribosome inactivating protein (RIP) plant toxin saporin 6. Cell protein synthesis inhibition assay reveals that the immunotoxins display a potent and specific cytotoxicity that is characterized by a slow rate, since the time required to inhibit incorporation of radiolabeled leucine completely ranges from 36 h to 60 h depending on the target cell line and the immunotoxin. Because this feature may hamper the immunotherapeutic use of these conjugates we analysed this further by studying the early phases of internalization of immunotoxins by immunoelectron microscopy. The results of this study have demonstrated that the distribution pattern of the immunotoxins and of the unconjugated mAb over the cell surface overlaps. Similarly the mAb and immunotoxins are internalized into the cell by two different pathways: via clathrin-coated pits or via smaller uncoated pits and vesicles. A higher degree of internalization is achieved when the two immunotoxins are used in combination. Unlike the slow kinetics of cell intoxication the process of immunotoxin endocytosis is characterized by a rapid rate of internalization (above 40% at 5 min in the SK-BR-3 cell line). Although these findings provide no clue to explain the mechanisms of the slow rate of cytotoxicity of the two immunotoxins their rapid internalization indicates that these reagents can be exploited in immunotherapeutic approches to gp185^HER-2-expressing malignancies.     

Internalizing Cancer Antibodies from Phage Libraries Selected on Tumor Cells and Yeast-Displayed Tumor Antigens

A number of approaches have been utilized to generate antibodies to cancer cell surface receptors that can be used as potential therapeutics. A number of these therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor but also undergo internalization into the cell upon binding. We previously reported on the ability to generate cancer cell binding and internalizing antibodies directly from human phage antibody libraries selected for internalization into cancer cell lines. While a number of useful antibodies have been generated using this approach, limitations include the inability to direct the selections to specific antigens and to identify the antigen bound by the antibodies. Here we show that these limitations can be overcome by using yeast-displayed antigens known to be associated with a cell type to select the phage antibody output after several rounds of selection on a mammalian cell line. We used this approach to generate several human phage antibodies to yeast-displayed EphA2 and CD44. The antibodies bound both yeast-displayed and mammalian cell surface antigens, and were endocytosed upon binding to mammalian cells. This approach is generalizable to many mammalian cell surface proteins, results in the generation of functional internalizing antibodies, and does not require antigen expression and purification for antibody generation.     

Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface–bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor–labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.     

Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells

We have recently identified and validated the prostate cancer antigen Tomoregulin as a target for the radioimmunotherapy for prostate cancer. Here, we provide evidence that Tomoregulin is an internalizing antigen and a potential target for immunotoxins. First, the cell surface localization of Tomoregulin was confirmed by flow cytometry, and its expression levels were determined by whole-cell binding assays. Second, laser scanning confocal microscopy revealed Tomoregulin internalization into the cytoplasm on antibody binding at 37 degrees C. The internalized Tomoregulin was found to colocalize with acidic vesicles. Third, internalization kinetics assays using (125)I-labeled anti-Tomoregulin mouse monoclonal antibody 2H8 demonstrated that the amount of internalized antigen-antibody complexes increased with time and reached approximately 25% of the total surface antigen after 60 to 90 minutes. Because 2H8 is capable of binding to Tomoregulin on the cell surface and can be internalized, we finally evaluated 2H8 as a means of targeting toxic payloads to prostate cancer cells. 2H8 was coupled to the cytotoxin saporin through a secondary antibody (Mab-ZAP) in indirect immunotoxin assays. Cell killing occurred on Tomoregulin-positive cells (Clone69) at the immunotoxin concentrations not affecting the Tomoregulin-negative cells (PC-3). In contrast to 2H8, the control antibody (mouse anti-c-Myc antibody 9E10) had no effect on cells in the presence of Mab-ZAP. Thus, Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells, and Tomoregulin-mediated delivery of immunotoxin has potential as a prostate cancer therapy.     

Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin

Hybrid molecules obtained through conjugation of monoclonal antibodies and toxins constitute an approach under exploration to generate potential agents for the treatment of cancer and other diseases. A frequently employed toxic component in the construction of such immunotoxins is ricin, a plant toxin which inhibits protein synthesis at ribosomal level and so requires to be internalized by the cell. A hemolytic toxin isolated from the sea anemone Stichodactyla helianthus, which is active at the cell membrane level, was linked through a disulfide bond to the anti-epidermal growth factor receptor monoclonal antibody ior egf/r3. The resulting immunotoxin did not exhibit hemolytic activity except under reducing conditions. It was toxic for H125 cells that express the human epidermal growth factor receptor, but non-toxic for U1906 cells that do not express this receptor.     

An immunogold-silver staining method for detection of cell-surface antigens in light microscopy

An immunogold-silver staining technique for detection of cell-surface antigens in cell suspensions was developed. Leukocyte cell suspensions were first incubated with monoclonal antibodies directed against cell-surface antigens and then with colloidal gold-labeled goat anti-mouse antibodies. Cytocentrifuge preparations of the cell suspensions were immersed in a physical developer containing silver lactate and hydroquinone as reducing substance. The preparations were then counterstained and mounted. In light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. An optimal morphology, as revealed by a May-Grünwald-Giemsa counterstain, permitted accurate cell identification. The labeling was influenced by the gold particle diameter and the concentration of the gold reagents, by the duration of incubation in the physical developer, and by the composition and temperature of this medium. The T-cell subsets enumerated with this method in the peripheral blood of normal adults were identical to those found with other methods. The sensitivity of the technique was comparable with that of immunofluorescence microscopy. This immunogold-silver staining procedure proved to be a reliable tool for detection of cell-surface antigens in light microscopy.     

Serotherapy of ovarian cancer

The development of monoclonal antibodies has permitted the identification of several ovarian-tumor-associated antigens which might serve as targets for serotherapy in vivo. With the exception of antibodies directed against growth factor receptors, unmodified monoclonal reagents must activate complement (C') components or bind effector cells to destroy tumor targets. Antibody-dependent cell-mediated cytotoxicity (ADCC) may be particularly important for eliminating tumor cells in vivo. A shortage of functionally active effector cells can limit the efficacy of serotherapy with heteroantisera or monoclonal reagents. The use of immunostimulants such as Corynebacterium parvum has increased the number and activity of effector cells for ADCC within the peritoneal compartment of mice and of patients with ovarian cancer. Intraperitoneal serotherapy can achieve direct contact between antibody and microscopic deposits of ovarian tumor cells which persist following cytoreductive operations and cytotoxic chemotherapy. Conjugation of monoclonal antibodies with radionuclides, drugs or toxins might increase the potency of serotherapy and circumvent the effector shortage. Clinical studies to date have evaluated radionuclide conjugates for imaging and for therapy. Patients with a small volume of disease have responded to treatment. Preclinical models suggest that drug and toxin conjugates might also prove active. Recent studies have demonstrated a synergistic interaction between different immunotoxins. Ovarian carcinoma is likely to be a valuable clinical model for evaluating immunoconjugates which react with epithelial tumor cells.     

Selective cytotoxicity of antibody-ricin-A-chain conjugate for human tumor B cells. II. Comparison of activity of immunotoxins conjugated with poly- and monoclonal antibodies

The A-chain of a plant toxin ricin has been coupled to poly- and monoclonal antibodies specific to the L-chains of human IgG. The inhibitory effect of the conjugates has been compared with the ability of the antibodies to bind to target cells. Cytotoxicity of the conjugates has been monitored following incorporation of 14C-leucine radioactivity into Burkitt lymphoma cells with surface Ig. The 50% inhibition of protein synthesis is observed 18 h after treatment of cells with immunotoxins, when the concentration of the conjugates with poly- and monoclonal antibodies is 1.2.10(-9) M and 0.7.10(-9) M, respectively. The data take into account that only part of the polyclonal antibodies molecules is able to react with target cells. The control conjugates containing either monoclonal antibodies that do not react with the lymphoma cells surface L-chains or nonimmune serum IgG proved to have no effect on target cells even at the level of 10(-7) M. The immunotoxins with poly- and monoclonal antibodies produce almost the same kinetics of protein synthesis inhibition, when incubated with lymphoma cells for 60 min. However, a 30 min treatment reveals a considerably higher cytotoxicity of the conjugate with monoclonal antibodies.     

Hybridomas: a new dimension in biological analyses

Since the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic increase in publications using these reagents has been in itself fascinating and informative. An overview of the development of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules, differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the regulation of growth control and cell division are provided.     

Atypical multidrug resistance in CCRF-CEM cells selected for high level methotrexate resistance: reactivity to monoclonal antibody C219 in the absence of P-glycoprotein expression

A series of CCRF-CEM sublines selected for extreme resistance to methotrexate has been shown previously to exhibit cross resistance to a number of agents belonging to the multidrug resistance phenotype (J.Natl.Cancer Inst.1989; 81, 1250-1254). The role of the mdr1 gene and its product (P-glycoprotein) in this atypical pattern of multidrug resistance has now been investigated. Southern and Northern analyses failed to demonstrate any amplification, rearrangement or over-expression of the mdr1 gene in the drug-resistant cells. Similarly, monoclonal antibodies MRK16 and JSB1 revealed no increase in the amount of P-glycoprotein present. By contrast, monoclonal antibody C219 detected a 170 kDa protein in all sublines, and in highest concentration in the most resistant cells. The results raise the possibility that a novel, C219-reactive protein may mediate resistance to both methotrexate and members of the multidrug resistance family.     

Unique Biological Properties of Catalytic Domain Directed Human Anti-CAIX Antibodies Discovered through Phage-Display Technology

Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.     

Detection of recombinant P-glycoprotein in multidrug resistant cultured cells

The MDR1 multidrug resistance gene encodes a high molecular weight membrane-spanning cell surface protein, P-glycoprotein, that confers multidrug resistance by pumping various cytotoxic drugs, including vinblastine, doxorubicin or paclitaxel, out of cells. Overexpression of P-glycoprotein in human tumors has been recognized as a major obstacle for successful chemotherapy of cancer. Thus, P-glycoprotein represents an important drug target for pharmacological chemosensitizers. Initially, cell culture models to study the multidrug resistance phenotype were established by selecting drug-sensitive cells in step-wise increasing, sublethal concentrations of chemotherapy agents. P-glycoprotein was found to be overexpressed in many of these models. Multidrug resistant cells can also be generated by transfection of cultured cells with the MDR1 gene, followed by selection with cytotoxic drug at a concentration that kills all untransfected host cells. Transfectants expressing wild-type or mutant recombinant P-glycoprotein have significantly contributed to our understanding of the structure of P-glycoprotein and its molecular and cellular functions. Additionally, the MDR1 gene has also been used as a selectable marker for the transfer and coexpression of non-selectable genes. This article details means for detection of P-glycoprotein in DNA-transfected or retrovirally transduced, cultured cells. Different experimental approaches are described that make use of specific antibodies for detection of P-glycoprotein. Strategies to visualize P-glycoprotein include metabolic labeling using ³⁵S-methionine, labeling with a radioactive photoaffinity analog, and non-radioactive immunostaining after Western blotting.     

The epitope specificity and tissue reactivity of four murine monoclonal anti-CD22 antibodies

The CD22 antigen is expressed on the surface of normal human B cells and some neoplastic B cell lines and tumors. Previous cross-blocking studies using a panel of monoclonal anti-CD22 antibodies have defined four epitope groups, termed A-D. In the present studies, we have further dissected the epitopes recognized by four monoclonal anti-CD22 antibodies using immunoprecipitation and cross-blocking techniques, immunofluorescence analyses with a variety of cell lines, and immunoperoxidase analyses of 36 normal human tissues. Two of the antibodies, HD6 and RFB4, have been described previously, and two, UV22-1 and UV22-2, are described in this report. Our studies indicate that the four monoclonal antibodies show unexpected complexities in their reactivity with CD22+ and CD22- cells and their reactivity with solubilized CD22 molecules. The four antibodies, which recognize epitopes defined previously as CD22-A and CD22-B, further subdivide these epitope clusters into four determinants, A1, A2, B1, and B2. Furthermore, only two of the antibodies, RFB4 and UV22-2, are B cell-specific. In summary, our data indicate that RFB4 and UV22-2 would be the antibodies of choice for constructing immunotoxins to treat B cell tumors.     

Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein

Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb). MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells. MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane. Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues. In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples. A similar pattern was found using MAb C219 in rat tissues. in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle fibers in rat and human tissues. ATPase cytochemistry showed that these reactive skeletal muscle fibers were of the type I (slow-twitch) class. Other additional sites of C219 reactivity in rat tissues were found in pancreatic acini, seminal vesicle, and testis. Electrophoretic gel immunoblotting showed two protein bands reactive with MAb C219. In liver, MAb C219 reacted with a approximately 170 KD band. In skeletal and cardiac muscle, MAb C219 reacted with a approximately 200 KD band which migrated in the same position as myosin. This band also reacted with an antibody to skeletal muscle myosin. This result suggests that C219 may crossreact with the heavy chain of muscle myosin in cardiac and skeletal muscle. Because MAb C219 reacts with proteins other than P170, it should be used with caution in studies of multidrug resistance.     

Comparison of multiple anti-CEA immunotoxins active against human adenocarcinoma cells

Summary Anti-carcinoembryonic antigen (CEA) immunotoxins constructed with multiple anti-CEA antibodies (goat and baboon polyclonal, and three murine monoclonal antibodies) by covalently linking them to the A chain of ricin via a disulfide bond all function as potent and specific toxins for CEA-bearing cells, suggesting that the CEA molecule is capable of directing productive internalization of ricin A chain. The high potency of anti-CEA immunotoxins apparently makes addition of ricin B chain unnecessary for high toxic efficiency, as in some other systems, because presence of the B chain reduces target cell specificity. Several characteristics of the immunotoxins which might account for their cytotoxic potency were studied. Equilibrium association constants of the goat, baboon, and murine monoclonal C-19 antibodies with fluid-phase CEA were determined by using Langmuir plots and were found to be 8.79, 6.61, and 8.13×10⁹ M ^–1, respectively, indicating the high and similar affinities of the three antibodies toward CEA. Radioimmunoassay binding studies of the three immunotoxins with ¹²⁵I-CEA showed that the antibody portions of the molecules retained the ability to form complexes with CEA after conjugation to ricin A chain. The maximum number of anti-CEA antibody molecules bound per cell, as demonstrated by ¹¹¹In-labeled C-19 binding assays with CEA-bearing cell lines, varied from 2.65×10⁵ per cell for HT29 to 2.01×10⁶ for LoVo, with an intermediate value of 1.17×10⁶ per cell for WiDr. Cytotoxicity of the immunotoxins was assessed by inhibition of protein synthesis and expressed as a median inhibitory dose (ID₅₀). Comparison of the ID₅₀'s of each immunotoxin on the three cell lines has shown that the immunotoxin made of the monoclonal C-19 antibody is in general 6 to 7 times more cytotoxic than the goat and baboon antibody immunotoxins. The affinity of CEA-antibody binding is probably an important, but not a sole factor in determining the immunotoxin potency. The fact that the antibodies with very similar affinity toward fluid phase CEA make immunotoxins of different potency might indicate that interactions with membrane-bound CEA are more complex and/or the efficiency of internalization of various immunotoxins is different. An important factor in immunotoxin action appears to be the CEA content in target adenocarcinoma cells.Supported in part by the NIH BRSG grant SO7RRO5712, the American Cancer Society, Mass. Div. Research Grant 1543-C-1, and by the Aid for Cancer Research (Boston) award to L. V. L., and by RO1 CA 29160 and RO1 CA 39748 grants to T. W. G.     

Detection of P-glycoprotein in human leukemias using monoclonal antibodies

Overexpression of a Mr 170,000 membrane glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines. Two monoclonal antibodies (Mab) against P-glycoprotein (265/F4 and C 219) were used to examine tumour samples from patients with leukemias for evidence of P-glycoprotein overexpression. High levels of P-glycoprotein (greater than 5% positive cells) were detected with both antibodies in samples from 3 out of 18 patients suggesting that a multidrug resistant phenotype may also occur in human leukemias.     

Evaluation of ricin A-chain immunotoxins directed against human T cells

We have synthesized four immunotoxins (ITs) by covalently coupling the A chain of ricin to murine monoclonal antibodies that recognize surface antigens on human T cells. Treatment of human peripheral blood lymphocytes with either 10.2-A, directed against the CD5 (Tp67) antigen, or 64.1-A, directed against the CD3 (Tp19) antigen, abolished protein synthesis in cells subsequently cultured with phytohemagglutinin (PHA). In contrast, two other ITs (9.6-A and 35.1-A), both directed against the CD2 (Tp50) antigen, had minimal effects on protein synthesis in PHA-stimulated cells. The binding of each IT to T cells was shown by immunofluorescence with fluorescein-conjugated goat anti-mouse immunoglobulin (FITC-GAMIg) and fluorescein-conjugated rabbit anti-ricin A-chain (FITC-RAR) antibodies. Activity of the ricin A chain in each IT was demonstrated by its ability to inhibit protein synthesis in a cell-free reticulocyte lysate assay. Ultrastructural immunoperoxidase analysis of IT internalization showed that ineffective and effective ITs were endocytosed at the same rate (50% of cells had labeled endosomes after 15 min). However, ineffective IT 35.1-A was more rapidly delivered to lysosomes (15-30 min) than effective ITs (10.2-A and 64.1-A) (greater than or equal to 30 min). The data support the hypothesis that there are several distinct pathways for internalization of ITs and that the ability of ricin A chain to reach and inactivate ribosomes may depend upon the specific membrane receptor involved in binding a given IT, its route of internalization, and the rate of entry of the IT into lysosomes.     

单抗LC-1与针对的人肺腺癌SPC-A-1细胞肿瘤相关抗原复合物的内化研究

我们运用抗人肺癌单抗LC-1结合胶体金免疫电镜技术研究了人肺腺癌SPC-A-1细胞表面抗原抗体复合物被内吞的全过程,发现该细胞表面抗原抗体复合物是通过受体介导内吞途径被内化,经多泡体富集后至溶酶体消化降解。此外我们还用流式细胞仪分析了内吞前后该细胞表面抗原量的变化和短期内的恢复情况。LC-1在诱发该细胞表面抗原内化的同时还诱发了它对该核糖体的自噬。     

Monoclonal antibodies: methods and clinical laboratory applications

Kohler and Milstein have shown that individual clones of normal antibody-secreting lymphocytes could be immortalized by fusion with myeloma cells. These investigators initiated a new era of technology with the successful in vitro production of monoclonal antibodies via somatic cell hybridization. With the use of monoclonal antibodies, many major problems arising from the limited specificity and reproducibility of conventional antisera can be solved. Some of the commonly employed methods for the production of monoclonal antibody are: (1) fusion of sensitized lymphocytes and myelomas from different sources to produce continuous antibody-producing cell lines; (2) in vitro viral transformation of sensitized lymphocytes to form continuous antibody-producing cells; (3) hybrid fusion of sensitized lymphocytes and continuous B lymphocyte cell lines. During the past few years, monoclonal antibody methodology has been used in almost every area of biological research. Monoclonal antibodies have been used as structural probes for proteins and hormones, and as highly specific agents for histocompatibility testing, tumor localization, immunotherapy, purification of molecules, identification of new surface antigens on lymphocytes and tumor cells, and detection of drug levels and microbial and parasitic diseases. In addition, several investigators have developed alternative methods for the production of human as well as mouse and rat monoclonal antibodies. The new technology of in vitro production of animal and human monoclonal antibodies will have many future applications in diagnosis and therapy in laboratory and clinical medicine.     

Identification of a 170 kDa membrane kinase with increased activity in KB-V1 multidrug resistant cells

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of ³²P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723–20729, 1989; Mol Cell Biol 10:3020–3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca⁺⁺, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr^R and murine melanoma B16/Adr^R. cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.