. 7'J #'/-7 - NASA · Report Period: Narrative: Monoclonal Antibodies Directed Against Surface...

NASA-CR-195102

Grant Number:

Semi-Annual Status Report

NASA-NAG 2-819

._7'J #'/-7 ©

Grant Period: 1/01/93 - 12/31/95

Principal Investigator: Andrew O. Martinez, Ph.D.

Institution: University of Texas at San Antonio

Project Title:

Report Period:

Narrative:

Monoclonal Antibodies Directed Against Surface

Molecules of MulticeU Spheroids

7/01/93 - _/31/93

The objective of this project is to generate a library of monoclonal antibodies (MAbs)

to surface molecules of mammahan tumor and transformed cells grown as

multicell spheroids (MCS). These MCS are highly organized, 3-dimensional

multicellular structures which exhibit many characteristics of in vivo organized

tissues not found in conventional monolayer or suspension culture; therefore, MCS

make better in vitro model systems to study the interactions of mammalian cells.

Additionally, they provide a functional assay for surface adhesion molecules.

The aims for this reporting period were: (1) to continue generating a library of

hybridomas producing antibodies against surface molecules of human and rodent

tumor and transformed cells grown as MCS using a subtractive immunization

scheme; and (2) to begin to characterize the binding patterns of selected MAbs from

our library on panels of human and rodent cell lines by immunofluorescence

microscopy and flow cytometry. To this end, six additional hybridomas producing

antibody of interest were identified and added to the library during the reporting

period. In addition, five hybridomas which were previously reported were cloned

and subcloned to ensure that they were producing monospecific antibody. We also

began to characterize the binding patterns of selected MAbs on panels of normal,

tumor and transformed human and rodent cell lines (see Appendix for list).

Prehminary results of these studies have identified three distinct monoclonal

antibody types: (1) MAbs that bind specifically to human cell lines; (2) MAbs that

bind specifically to rodent cell lines; and (3) MAbs that bind to both human and

rodent cell lines. Moreover, in some cases there were differences in binding patternand binding intensity between tumor/transformed and normal cell lines. (These

data have been summarized and are presented in the Appendix). This observation

is of great interest since the differences may reflect molecules which may be

expressed differentially in tumor/transformed and normal cells. Moreover, these

molecules could be the ones responsible for the abnormal behavior (aggregation,

(NASA-CR-I 95102) MCNOCLONAL

ANTIBF}DIES OIRECTED AGAINST SURFACE

MOLECULES OF MULTICELL SPHEROIDS

Semiannua| Status Report, i Jul. -

31 Dec. 1993 (Texas Univ.) 33 p

N96-23633

Unclas

https://ntrs.nasa.gov/search.jsp?R=19940019160 2019-12-28T07:54:23+00:00Z

compaction, 3-dimensional growth) exhibited by tumor and transformed cells.

The specific aims for the next six-month period are: (1) to continue to expand our

MAb library; (2) to continue to characterize the binding pattern and bindingintensity of selected MAbs on panels of human and rodent cell lines; and (3) to

initiate functional biological assays to determine whether MAbs can modulate the

cell-cell interactions (aggregation, compaction) of normal, tumor and transformedcells.

This project also supports the research training of two underrepresented minority

students (one graduate and one undergraduate). Both students made significant

contributions to the work accomplished during the reporting period. Moreover,

each student submitted an abstract and presented a paper at a national scientific

meeting (copy included in Appendix ). One student was accepted to the Universityof Texas Medical School at Houston and will start in fall 1994.

Appendix

Tissue Culture Cell Lines

WI38sv40

WI38

Hela 6TG r-

IMR90sv40

IMR90

GM3320

Hep3b

2DF*F 1

B14150

CHO 77256

FTO-2B

Mouse L

human sv40 transformed lung fibroblast

normal human lung fibroblast

human cervical carcinoma (6TG resistant)

human sv40 transformed lung fibroblast

normal human lung fibroblast

human neuroblastoma

human hepatoma

Con A resistant mutant (of B14150)

Chinese hamster peritoneal

Chinese hamster ovary

rat liver cells: TK-

mouse fibroblast

Summary of monoclonal antibody 5A12 (WSJ-2) by fluorescence

microscopy.

CELL LINE FLUORESCENCE INTENSITY

FIXED LIVE

WI38sv40 _ +++

Hela 6TG r- +t- +++

IMR90sv40 4+ I.P.

GM3320 -H- I.P.

Hep3b _ I.P.

B14150 + I.P.

2DF*F1 + +

P_AZT.F..R_

cell-cell

cell-surface

cell-surface with cell-

cell

cytoplasmic

around edges & cellsurface

granular cytoplasmic

weak "all over"

CHO 77256

FTO-2B

Mouse L

++ I.P.

+t+ I.P.

cytoplasmic


I.P. = in progress

FLUORESCENCE INTENSITY

- = no fluorescence

+ = weak

++ = medium

+++ = strong

+4++ = very strong

ANTIBODY $A12 : This antibody has been renamed WSJ-2

and is a Kappa IgG1 isotype which

exhibits a cell- cell binding patternon WI38sv40 cells.

CELL LINE

WI38sv40

WI38

Hela 6TGr

2DF*F1

B14150

FLOW CYTOMETRY

*Net Fluorescence

+t+ (333.27)

+_ (236.02)

+ (173.41)

*NET FLUORESCENCE- =<50

+ => 1004+ =>200

+++ =>300

++4+ =>400

*Net fluorescence values were obtained by subtracting the meanfluorescence value of the negative control, from the mean fluorescence

value of the monoclonal antibody being tested. (Mean fluorescence values

wer convertd from log to linear prior to calculations (logl0 X 256) ).

Summary of monoclonal antibody 10D8 (WSJ-3) by fluorescence

microscopy.


FIXED L I V E

WI38sv40 +t+ +++

Hela 6TG r- _ +++

IMR90sv40 +¢+ I.P.

GM3320 -¢+ I.P.

Hep3b + I.P.

B14150

2DF*F1

CHO 77256

Hep3b

FTO-2B

Mouse L

+ I.P.

-14- +

+++ I.P.

+ I.P.

+ I.P.

PATTERN

cell-cell

intermittant


cell

cytoplasmic

around edges of cell

cell-surface, some cell-

cell

cell-surface & between

cells

cytoplasmic

around edges of cell


I.P. = in progress

+

-H-

.+-I-I-

++++ =

FLUORF__CENCE INTENSITY

= no fluorescence

= weak

= medium

= strong

very strong

ANTIBODY 10D8 ; This antibody has been renamed WSJ-3


exhibits a cell- cell binding patternon WI38sv40 cells.

CELL LINE

WI38sv40

WI38

Hela 6TGr

2DF*F1

B14150

FLOW CYTOMETRY

*Net Fluorescence

+t+ (308.67)

-H- (210.45)

+ (166.44)

*NET FLUORESCENCE

- =<50

+ =>100

4+ =>200

=> 300

++++ =>400

*Net fluorescence values were obtained by subtracting the meanfluorescence value of the negative control, from the mean fluorescence



.Summary of monoclonal antibody 8El0 (WSJ-4) by fluorescence

microscopy.

CELL LINE FLUORESENCE INTENSITYFIXED L I V E

WI38sv40 +t+ +++

Hela 6TG r- -_+ +++

IMR90sv40 -t+ I.P.

GM3320 -t+ I.P.

Hep3b +t+ I.P.

B14150 + I.P.

2DF*F1 - -

CHO 77256 ++ I.P.

FTO-2B -t++ I.P.

Mouse L - -

PATTERN

cell-cell

cell surface


cell

cytoplasmic

around edges & cellsurface


cytoplasmic


I.P. = in progress

FLUORF_CENCE INTENSITY- = no fluorescence

+ = weak

++ = medium

+++ = strong

= very strong

ANTIBODY 8E10 ; This antibody has been renamed WSJ-4 and is

a Kappa IgG1 isotype which exhibits a cell-cell

binding pattern on WI38sv40 cells.

CELL LINE

WI38sv40

WI38

Hela 6TGr

2DF*F1

B14150

FLOW CYTOMETRY

*Net Fluorescence

+++ (316.99)

_- (219.70)

+ (165.66)

*NET FLUORESCENCE

- =<50

+ => 100

-t+ => 200

-H-+ => 300

++-1+ =>400

*Net fluorescence values were obtained by subtracting the mean

fluorescence value of the negative control, from the mean fluorescence



Summary of monoclonal antibody 6A3 (WSJ-5) by fluorescencemicroscopy.


FIXED LIVE PATTERN

WI38sv40 +++ +++

Hela 6TG r- 4+ +++

IMR90sv40 44+ I.P.

GM3320 _ I.P.

Hep3b - I.P.

B14150 - I.P.

2DF*F1 - -

CHO 77256 - I.P.

FTO-2B I.P.

Mouse L

cell-surface

cell-surface

cell-surface

granular cell-surface

cytoplasmic


I.P. = in progress

FLUORESCENCE INTENSITY- = no fluorescence

+ = weak

++ = medium

= strong

++-t+ = very strong

ANTIBODY 6A3 : This antibody has been renamed WSJ-5 and is

a Kappa IgG1 isotype which exhibits a cell-

surface binding pattern on WI38sv40 cells.

CELL LINE

WI38sv40

WI38

Hela 6TGr

2DF*F1

B14150

FLOW CYTOMETRY

*Net Fluorescencg

(367.87)

.t++ (390.22)

++++ (401.68)

*NET FLUORESCEN(_=<50

+ => 100

=>200

=>300

++_ =>400





Summary of monoclonal antibody 3C3 (WSJ-6) by fluorescence

microscopy.


WI38sv40

Hela 6TG r-

IMR90sv40

GM3320

Hep3b

B14150

2Dr*F1

CHO 77256

FTO-2B

Mouse L

FIXED

44+

+t-

-t-,t-t+

L I V E PATTERN

+++ cell-surface

+++ cell-surface

I.P. cell-surface

I.P.

I.P.

I.P.

I.P. = in progress

FLUORF_CENCE INTENSITY

= no fluorescence

+ -- weak

++ = medium

+++ = strong

++++ = very strong

ANTIBODY 3C3 ; This antibody has been renamed WSJ-6 and is

a Kappa IgG1 isotype which exhibits a cell-

surface binding pattern on WI38sv40 cells.

CELL LINE

WI38sv40

WI38

Hela 6TGr

2DF*F1

B14150

FLOW CYTOMETRy

*Net Fluorescence

(413.83)

++_ (423.81)

++++ (452.11)

*NET FLUORF__CENCE- =<50

+ => 100

=> 200

+++ =>300

+t-t-I- =>400





Summary of monoclonjll antibody DF1.2 (BDFI.2) by fluorescencemicroscopy.

CELL LINE

B14150

FLUORESCENCE

FIXED LIVE

+-IF

2DF*F1 +H- +++

CHO 77256 +++ I.P.

FTO-2B + I.P.

WI38sv40 + I.P

Hela 6TG r- I.P.

IMR90sv40 - I.P.

GM3320 +/- I.P.

INTENSITY

PATTERN

cell-surface & cell-

cell

cell-surface & between

cells

cell-surface;some cellto cell

weak cell surface

weak cell surface

n/a

n/a

binds only a

subpopulation of cells;between cells

I.P. = in progress

+

+/-

-1-+

4-t+

4-+4-+ =


= no fluorescence

= weak

= subpop= medium

= strong

very strong

ANTI.BODY DF1,2 ; This antibody has been renamed BDF1.2


exhibits a cell- cell binding patternon 2DF*F1 and B14150 cells.

CELL LINE FLOW CYTOMETRY*Net Fluorescence

2DF*FI ++++ (524.30)

B14150 ++++ (515.35)

WI38sv40 - (-6.6)

Hela 6TGr - (- 3.65 )

*NET FLUORESCENCE

=<50

+ =>50

-I+ =>200

+++ => 300

+'H'+ =>400





Summary of monoclonal antibody DF4.2 (BDF4.2) by fluorescence

microscopy.

CELL LINE

B14150

2DF*F1

CHO 77256

FLUORESCENCE

FIXED LIVE

INTENSITY

P_&Z.T.KI

- n/a

+++ speckled/cell-cell

I.P. cell-surface;some cellto cell

I.P. n/a

I.P n/a

I.P. n/a

I.P. n/a

I.P. n/a

-H-l-

+4-+

FTO-2B

WI38sv40

Hela 6TG r-

IMR90sv40

GM3320

I.P. = in progress


+ = weak

+/- = subpop++ = medium

+++ = strong

++++ = very strong

ANTIBODY DF4,2 ; This antibody has been renamed BDF4.2


exhibits a fragmented (speckled)

binding pattern including localization tointercellular boundaries and

extracellular matrix.

CELL LINE

2DF*F1

B14150

WI38sv40

Hela 6TGr

FLOW CYTOMETRY*Net Fluorescence

+ (185.8)

- (2.85)

- (-6.6)

- (-3.65)

*NET FLUORESCENCE

- =< 50

+ =>50

4+ => 200

+++ =>300

-t-t-t+ =>400





Summary of monoclonal antibody 10HI0 (BDF7.1) by fluorescence

microscopy.

CELL LINE

B14150


FIXED LIVE

+ or- +/- cell surface; some cell-cell

+++ +++ speckled/cell-cell

+++ I.P. speckled/cell-cell

- I.P. n/a

- I.P n/a

- I.P. n/a

- I.P. n/a

+/- I.P. n/a

2DF*F1

CHO 77256

FTO-2B

WI38sv40

Hela 6TG r-

IMR90sv40

GM3320

I.P. = in progress

FLUORESCENCEINTENSITY= no fluorescence

+ = weak


+++ = strong

++++ = very strong+ or - = unstable

ANTIBODY DF10H10 ; This antibody has been re,named BDF7.1


exhibits a fragmented (speckled)

binding pattern including localization tointercellular boundaries and

extracellular matrix.

CELL LINE

2DF*F1

B14150

WI38sv40

Hela 6TGr

FLOW CYTOMETRY*Net Fluorescence

(10.05)

(10.0)

+ (159.15)

+ (71.75)

*NET FLUORESCENCE

- =<50

+ =>50

+1- => 200

=>300

++++ =>400





Summary of monoclonal antibody DF3.2 (B_.2) by fluorescence

microscopy.


FIXED LIVE

B14150 n/a

2DF*F1 +_ +++ speckled/cell-cell

CHO 77256 +++ I.P. speckled/cell-cell

FTO-2B + I.P. cell surface

WI38sv40 + I.P cell surface

Hela 6TG r- + I.P. cell surface

IMR90sv40 + I.P. cell surface

GM3320 + I.P. cell surface

I.P. = in progress


+ = weak


= strong

++++ = very strong

Summary of monoclonal antil_ody 2Gll (BDF5.1) by fluorescence

microscopy.

CELL LINE FLUORESQENCE INTENSITY

FIXED LIVE

B14150

2DF*F1 ++++ ++++

CHO 77256 +++ I.P.

FTO-2B + I.P.

WI38sv40 - I.P

Hela 6TG r- - I.P.

IMR90sv40 - I.P.

GM3320 - I.P.

n/a

speckled/cell-cell

speckled/cell surface

cell surface

n/a

n/a

n/a

n/a

I.P. = in progress


- = no fluorescence

+ = weak

+/- = subpop

++ = medium

+++ = strong

++++ = very strong

Summary of monoclonal antibody 9C6 (BDF6.1) by fluorescencemicroscopy.

CELL LINE FLUORESCENCE INTENSITy

FIXED LIVE P.A.T.ZER_

B14150 - - n/a

2DF*FI _ +++ speckled/cell-cell

CHO 77256 +++ I.P. speckled/cell surface

FTO-2B + I.P. cell surface

WI38sv40 I.P n/a

Hela 6TG r- I.P. n / a

IMR90sv40 - I.P. n/a

GM3320 - I.P. n / a

I.P. = in progress


- = no fluorescence

+ = weak


= strong

++++ = very strong

BECTON

DICKINSON

DATE: 30-DEC-93

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m-r %aTn 4:30:24

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RELATIVE FLUORESCENCE INTENSITY

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DATE: 30-DEC-93

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DATE: 29-DEC-93

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DICKINSON

DA_E : 2-JAN-94

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DATZ: 2-JAN-94

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DICKINSON LYSYS _ Version 1C II/_°_

DATE: 3-JAN- 94 __-_.-.: 1:29:16

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DATE: 3-JAN-94 .x_.._: 1:42:28

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I-J

ISOLATION OF MONOCLONAL ANTIBODIES TO SURFACE MOLECULES OF A

COMPACTION MUTANT OF THE MULTICELL SPHEROID PHENOTYPE

We are using multicell spheroids (MCS) to study cell-cell

interactions of transformed cells. MCS are formed via cellular processes

(i.e., aggregation and compaction) mediated by surface molecules. We have

produced several mutants of B14150 (hamster) cells which are defective

in MCS formation. In this study, we have used a subtractive immunization

technique to isolate monoclonal antibodies (MAbs) to surface molecules of

a compaction-defective mutant, ConAR-2DF-FI. Initial screening by

fluorescence microscopy revealed two major types of MAb-surface binding

patterns. One type of MAb preferentially localized to intercellular

boundaries, while a second type exhibited a more general staining pattern

including localization to intercellular boundaries and extracellular matrix.

Studies are now in progress to determine whether these MAbs block MCS

formation of wildtype B14150 cells. (Supported by NSF (RII-9005546),

NASA (NAG 2-819) and NIH (GM 08194) grants).

Name:

Institution:

Classification:

Area of Study:

NSF Program:

Faculty Advisor:

Presentation Setting:

Sandra N. Garcia

University of Texas at San Antonio

Undergraduate - Senior

Cell BiologyRIMI

Andrew O. Martinez/(210) 691-4476Panel

C MONOCLONAL ANTIBODIES TO MULTICELL

SPHEROIDS OF HUMAN TRANSFORMED

CELLS USING A SUBTRACTIVE

IMMUNIZATION TECHNIQUE. CG Cantu, LA

Jordan, LS Armstrong, At Martinez, "llle University

of Texas at San Antonio, Division of Life Sciences,

San Antonio, "IX 78230.

Multicell tumor spheroids (MTS) are three-

dimensional in vitro models used in our laboratory

to study cellular and molecular mechanisms involvedin cell-cell interactions. MTS are formed via a two-

step process involving aggregation and compaction

mediated by surface molecules of tumor and

transformed cells. In this study, we are using a

subtractive immunization technique to increase

specificity to surface molecules involved in cell-cell

interactions. This scheme involved multiple

injections of WI38SV40 single cells (tolerogen) each

followed by injection of the immunosuppression

drug cyclophosphamide. Multiple injections of

WI38SV40 spheroids (immunogen) were then given,

theoretically generating antibodies that recognize the

molecules present on the immunogen that are not

present on the tolerogen. TIffs technique lms allowedus to isolate an increased number of MAbs that

exhibit binding patterns at points of cell-cell contactby florescence microscopy. The MAbs will be

further characterized by functional assays and flow

cytometry. Future studies will utilize the MAbs to

identify surface adhesion molecules involved in

MTS formation by Western blot analysis. (Supported

by NIH (GM 08194), and NASA (NAG 2-819)

grants, The University of Texas at San Antonio)

J

p •

_AI.. PAGE i_

POOR QUALITY

•11 r"

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